Curcumin-mediated antimicrobial photodynamic therapy prevents Candida biofilm formation in a species-dependent manner.

Biofilm-forming capacity was evaluated by quantifying total adherent biomass using the crystal violet assay, which enabled the classification of each Candida strain according to its biofilm production profile, as shown in Fig.  2 . Fig. 2 Total biomass (OD 620nm ) of Candida biofilms.  OD 620nm  Optical density at 620 nm, ATCC  American type culture collection Total biomass (OD 620nm ) of Candida biofilms.  OD 620nm  Optical density at 620 nm, ATCC  American type culture collection All yeasts evaluated formed biofilm; 4 were classified as strong biofilm producers. C. metapsilosis ATCC 96,143 showed the highest OD 620nm (0.478 ± 0.191), while C. dubliniensis ATCC MYA-646 presented the lowest OD 620nm (0.292 ± 0.129) and was the only yeast classified as a moderate biofilm producer. The other yeasts were classified as strong producers: C. albicans ATCC 10,231 (0.371 ± 0.163), C. orthopsilosis ATCC 96,141 (0.455 ± 0.149), and C. parapsilosis ATCC 22,019 (0.384 ± 0.125). Previous studies have reported different biofilm formation values for C. albicans , depending on the origin and evaluation method employed. One study reported OD 590nm between 2.410 and 2.530 for C. albicans ATCC 10,231 biofilms [ 20 ], with results differing from those obtained in our study (OD 620nm = 0.371 ± 0.163). This divergence should not be interpreted as biological inconsistency alone, since crystal violet quantification is highly sensitive to methodological variables, including inoculum density, incubation time, washing stringency, dye retention, destaining solvent, and optical wavelength. Another study analyzed clinical samples of C. albicans and C. dubliniensis isolated from sputum of cystic fibrosis patients and recorded OD 570nm between 0.280 and 0.640 for C. albicans and a higher value for C. dubliniensis (OD 570nm = 1.780) [ 15 ]. Although our results for C. albicans fall within this range, it is important to note that direct comparison is limited due to differences in isolates and methodology used in each experiment. Furthermore, our study used ATCC standard strains, unlike the other work. C. metapsilosis ATCC 96,143, C. orthopsilosis ATCC 96,141, and C. parapsilosis ATCC 22,019 were previously classified as having “weak biofilm formation” [ 28 ]. In another previous study, the biofilm of different Candida species, including strains of C. albicans , C. parapsilosis , C. orthopsilosis , C. metapsilosis , and C. parapsilosis ATCC 22,019, was analyzed, and the microorganisms showed OD 595nm = 1.761 ± 0.412 [ 18 ]. Together, these discrepancies reinforce that biofilm classification is not an intrinsic and fixed property of the strain, but rather an experimentally dependent phenotype strongly influenced by assay conditions. C. dubliniensis ATCC MYA-646 exhibited biofilm formation with OD 620nm = 0.542 ± 0.108 [ 2 ]. In our study, the same strain showed OD 620nm = 0.292 ± 0.129 and was classified as moderate. These variations reinforce the need for standardization of protocols for fungal biofilm quantification to ensure greater reproducibility among studies. Fungal biofilms exhibit intrinsic resistance to conventional antifungal treatments and host immune responses, making biofilm-related infections a major clinical challenge [ 13 ]. Additionally, antifungal resistance is complex and can be caused by several factors, including a response to a compound or an irreversible genetic alteration resulting from prolonged exposure to an antimicrobial [ 22 ]. Therefore, the confirmation that all tested strains formed measurable biofilms provides the experimental basis for evaluating curcumin-mediated antimicrobial photodynamic therapy against both early biofilm establishment and mature biofilm architecture. Curcumin-mediated aPDT inhibited biofilm formation in all analyzed yeasts. The best inhibition results were observed for C. metapsilosis ATCC 96,143 (86.82%) and C. parapsilosis ATCC 22,019 (83.33%) compared to control groups. The lowest reduction in biofilm formation was observed in C. albicans ATCC 10,231 (68.19%), while C. dubliniensis ATCC MYA-646 and C. orthopsilosis ATCC 96,141 were inhibited by 75.34% and 85.05%, respectively (Table  1 ). Table 1 OD 620nm of biofilms formed after curcumin-mediated photodynamic therapy and non-photoactivated controls Candida Curcumin (320 µM) Non- photoactivated controls Reductions Mean SD Mean SD % C. albicans ATCC 10,231 0.118 ± 0.029 0.371 ± 0.163 68.19 C. metapsilosis ATCC 96,143 0.063 ± 0.009 0.478 ± 0.191 86.82 C. dubliniensis ATCC MYA-646 0.072 ± 0.013 0.292 ± 0.129 75.34 C. orthopsilosis ATCC 96,141 0.068 ± 0.018 0.455 ± 0.149 85.05 C. parapsilosis ATCC 22,019 0.064 ± 0.009 0.384 ± 0.125 83.33 Non inoculated control 0.056 ± 0.004 0.055 ± 0.002 - SD Standard Deviation, ATCC  American Type Culture Collection OD 620nm of biofilms formed after curcumin-mediated photodynamic therapy and non-photoactivated controls SD Standard Deviation, ATCC  American Type Culture Collection Because the biofilm formation data did not meet the assumption of normality according to the Shapiro–Wilk test ( p  = 0.007), intergroup comparisons were performed using the Kruskal–Wallis test followed by Dunn’s multiple-comparison post hoc test. This analysis showed statistically significant reductions in biomass for all curcumin-mediated aPDT-treated groups compared with their respective non-photoactivated controls ( p  < 0.05), indicating that curcumin-mediated aPDT effectively interfered with the early stages of biofilm establishment across all tested Candida species. Importantly, the treated groups displayed OD 620nm values close to the non-inoculated control, particularly for C. metapsilosis , C. orthopsilosis , and C. parapsilosis , suggesting substantial impairment of initial adhesion and/or early biomass accumulation. Therefore, the preventive antibiofilm effect of curcumin-mediated aPDT was not only biologically evident but also quantitatively supported across the evaluated species. The light-only control showed that blue LED irradiation alone did not inhibit Candida biofilm formation under the tested conditions. In the absence of curcumin, all irradiated control groups maintained substantial biofilm biomass, with OD 620nm values of 0.371 ± 0.163 for C. albicans , 0.478 ± 0.191 for C. metapsilosis , 0.292 ± 0.129 for C. dubliniensis , 0.455 ± 0.149 for C. orthopsilosis , and 0.384 ± 0.125 for C. parapsilosis . These results confirm that the preventive antibiofilm effect was driven by curcumin photoactivation rather than by blue light alone. Previously, complete inactivation of planktonic yeast viability by curcumin-mediated aPDT (20 µM) was observed after 1 min of pre-irradiation against Candida dubliniensis CBS 7987 and Candida albicans ATCC 90,028, with curcumin prepared in 10% dimethyl sulfoxide (DMSO) and an LED device with 22.0 mW/cm² light intensity and 455 nm wavelength [ 3 ]. In other studies, curcumin demonstrated antifungal activity against clinical isolates of C. albicans and C. parapsilosis , with Minimum Inhibitory Concentrations (MICs) of 32 and 64 µg/mL, respectively [ 29 ]. Photoactivated curcumin with a 460 nm laser was active against C. albicans ATCC 10,231 and reduced viable yeast counts by 95% [ 7 ]. In our study, in addition to using different yeasts, we employed an LED device with lower light intensity (0.02165 W/cm²) and a wavelength of 450 nm. Therefore, the variability among these parameters may explain the differences observed between studies. Photoactivated curcumin exerts its antifungal action predominantly through the generation of ROS, which induce generalized oxidative damage to cells and the extracellular matrix of the biofilm [ 12 ]. During early biofilm development, cells are more exposed and the extracellular matrix is still immature, which may facilitate photosensitizer access, oxygen diffusion, and ROS-mediated disruption of adhesion and microcolony formation. This may explain why curcumin-mediated aPDT was more effective in preventing biofilm formation than in disrupting mature biofilms [ 31 ]. Membrane destabilization by curcumin facilitates its internalization, intensifying the photodynamic effect. Intracellular ROS oxidize proteins, lipids, and nucleic acids, causing DNA damage and protein inactivation, leading to metabolic paralysis and cell death [ 12 , 31 ]. Overall, these results indicate that curcumin-mediated aPDT is particularly effective as a preventive antibiofilm strategy, acting during the early stages of Candida biofilm development rather than primarily as a biofilm-eradicating intervention. The efficacy of curcumin-mediated aPDT against mature yeast biofilms exhibited marked species-dependent variability (Table  2 ). The highest reduction in biofilm biomass was observed for C. parapsilosis ATCC 22,019 (49.63%), whereas C. dubliniensis ATCC MYA-646 showed minimal susceptibility, with only 0.64% inhibition. In the mature biofilm assay, data distribution was compatible with normality according to the Shapiro-Wilk test ( p  = 0.284). Nevertheless, due to the limited sample size and to preserve a conservative statistical framework comparable to that used for the biofilm formation assay, non-parametric analysis was maintained. The Kruskal–Wallis test showed no statistically significant differences between curcumin-mediated aPDT-treated mature biofilms and their corresponding controls ( p  > 0.05). Consequently, although reductions of up to 49.63% were observed, particularly for C. parapsilosis ATCC 22,019, these effects should be interpreted as species-dependent tendencies rather than statistically validated mature-biofilm disruption. Table 2 OD 620nm of mature biofilms after curcumin-mediated phototherapy and non-photoactivated controls Candida Curcumin (320 µM) Non- photoactivated controls Reductions Mean SD Mean SD % C. albicans ATCC 10,231 0.270 ± 0.045 0.332 ± 0.063 18.67 C. metapsilosis ATCC 96,143 0.681 ± 0.147 0.707 ± 0.103 3.68 C. dubliniensis ATCC MYA-646 0.617 ± 0.105 0.621 ± 0.167 0.64 C. orthopsilosis ATCC 96,141 0.495 ± 0.093 0.581 ± 0.133 14.80 C. parapsilosis ATCC 22,019 0.275 ± 0.065 0.546 ± 0.141 49.63 Non inoculated control 0.058 ± 0.003 0.057 ± 0.003 - SD Standard Deviation, ATCC  American Type Culture Collection OD 620nm of mature biofilms after curcumin-mediated phototherapy and non-photoactivated controls SD Standard Deviation, ATCC  American Type Culture Collection The low response observed for C. dubliniensis may be associated with intrinsic properties of mature biofilms, particularly the extracellular polymeric matrix, which limits photosensitizer penetration and ROS diffusion, as well as the presence of metabolically heterogeneous and persister cell populations, known to confer tolerance to oxidative stress [ 23 ]. In addition, species-specific differences in stress response mechanisms may further contribute to reduced susceptibility to aPDT [ 6 ]. These findings reinforce the role of biofilm maturity as a critical determinant of aPDT efficacy. Unlike early biofilms, mature biofilms possess a consolidated three-dimensional structure, increased extracellular matrix density, altered metabolic gradients, and subpopulations of cells with reduced susceptibility to oxidative damage. Moreover, for mature biofilms, blue LED irradiation alone also did not substantially reduce established Candida biomass. The light-only controls showed OD620nm values of 0.332 ± 0.063 for C. albicans , 0.707 ± 0.103 for C. metapsilosis , 0.621 ± 0.167 for C. dubliniensis , 0.581 ± 0.133 for C. orthopsilosis , and 0.546 ± 0.141 for C. parapsilosis . Thus, although curcumin-mediated aPDT produced species-dependent reductions in mature biofilms, the light-only controls confirmed that irradiation alone was insufficient to disrupt established biofilm biomass. Mature biofilms represent a significant therapeutic challenge, as their mature structure confers intrinsic resistance to antimicrobial agents through multiple mechanisms, such as physical barrier, metabolic heterogeneity, phenotypic alterations, and persistent cells [ 32 ]. Divergent results have been observed in previous studies evaluating the efficacy of antimicrobial photodynamic therapy against Candida biofilms compared to our findings. For instance, Ma et al., [ 17 ] irradiated C. albicans biofilms (CCA1, CCA2, and ATCC 90028) with LED (455 nm, 22.0 mW/cm²) for 6 min. This resulted in viability inhibition of 90.87% for ATCC 90,028, 66.44% for CCA1, and 86.74% for CCA2. Another in vivo study investigated the application of a curcumin solution (10 mL, 7.5 mg curcumin) sprayed into the oral cavity of oncology patients suffering from oral mucositis. After four weekly applications over one month, a reduction in colony-forming unit counts was observed in the later evaluations (21 and 30 days). Andrade et al., [ 4 ]. Although the results of previous studies evidenced that photoactivated curcumin was active against yeast biofilms, in our study the activity was more discrete, with inhibition values not exceeding 50% of the total biofilm biomass. Methodological differences between the studies, such as LED light intensity, experimental phototherapy evaluation models, and different strains analyzed, may have influenced the results. Thus, the present findings reveal an important limitation of curcumin-mediated aPDT: while it strongly suppresses biofilm formation, its capacity to reduce pre-established mature biomass is more restricted and species dependent. This distinction is biologically relevant because mature biofilms are characterized by reduced photosensitizer penetration, limited oxygen availability in deeper layers, and increased tolerance to ROS-mediated injury [ 13 , 16 ]. In addition to damaging fungal cells, reactive oxygen species (ROS) in biofilms also break down polysaccharides and other components of the extracellular matrix. This degradation compromises the biofilm’s three-dimensional structure and resistance [ 14 ]. The internalization of curcumin by cells in both growth forms, planktonic and biofilm, is documented and constitutes a determining factor for treatment efficiency, allowing the generation of ROS to occur in critical intracellular compartments [ 11 , 26 ]. A thorough understanding of strategies to inhibit biofilm formation and to act against pre-formed biofilms is crucial for managing persistent infections and combating antimicrobial resistance [ 27 , 30 ]. In this regard, aPDT offers a promising approach for combating Candida biofilm formation, especially in medical device-related infections. The absence of relevant antibiofilm activity in the non-photoactivated controls supports the light-dependent nature of curcumin activity under the tested conditions. This finding indicates that photoactivation is required to enhance curcumin-mediated antibiofilm performance, consistent with the photochemical basis of aPDT. Mechanistically, this effect is interpreted in light of the well-established principles of aPDT. Upon irradiation, curcumin is excited from the ground state to an excited singlet state and subsequently undergoes intersystem crossing to a triplet state, as described by the Jablonski diagram [ 8 ]. This excited state enables interaction with O 2 via Type I and Type II photochemical pathways, leading to the generation of ROS, which are widely recognized as the primary mediators of microbial inactivation in aPDT. aPDT is a promising therapeutic strategy for combating microbial infections mediated by biofilms, particularly those caused by antimicrobial-resistant microorganisms. Given the current rise in infections that are resistant to conventional treatments, there is an increasing need for innovative approaches [ 19 ]. In this context, the present study supports curcumin-mediated aPDT primarily as a preventive antibiofilm strategy against Candida spp., while also indicating that optimization of irradiation parameters, photosensitizer delivery, and treatment protocols will be necessary to improve efficacy against mature biofilms.

This study examined the effectiveness of curcumin, when photoactivated by 450 nm LED light, against biofilms of five Candida species. We investigated its ability to both inhibit biofilm formation (preventive approach) and eradicate existing mature biofilms. Our findings indicate that photoactivated curcumin significantly inhibited biofilm development across all standard ATCC strains tested. Notably, C. metapsilosis and C. parapsilosis exhibited the highest susceptibility, with 86.72% and 83.26% inhibition, respectively. This inhibitory effect is likely associated with the reactive oxygen species (ROS) generated during photoactivation, which causes irreversible oxidative damage to vital cellular components, thereby impeding initial adhesion and the formation of the polymeric matrix. While aPDT demonstrated species-dependent activity against 48-hour mature biofilms, its efficacy was significantly attenuated. C. parapsilosis showed the most substantial biomass reduction at 49.63%, whereas C. dubliniensis exhibited the lowest efficacy, with only a 0.64% reduction. This disparity suggests that the mature biofilm’s architecture acts as a physical barrier, impeding photosensitizer penetration and reactive oxygen species (ROS) diffusion. Furthermore, the presence of metabolically heterogeneous and persistent cell populations within the mature biofilm may contribute to its resistance. Curcumin-mediated aPDT showed a strong inhibitory effect on biofilm formation, indicating significant preventive potential. However, its efficacy against mature biofilms was limited and species-dependent, reflecting the intrinsic resistance of established biofilm structures. These findings indicate that aPDT is more effective as a preventive strategy rather than for the eradication of mature biofilms, without implying a direct evaluation of intermediate developmental stages. Therefore, optimizing photodynamic parameters and developing synergistic strategies to enhance photosensitizer penetration and overcome biofilm-associated resistance are essential to improve its therapeutic applicability.

Five standard strains of Candida yeasts were evaluated: C. albicans ATCC 10,231, C. metapsilosis ATCC 96,143, C. dubliniensis ATCC MYA-646, C. orthopsilosis ATCC 96,141, and C. parapsilosis ATCC 22,019. The curcumin stock solution was obtained by weighing 16.2 mg of synthetic curcumin (98%) (PDT Pharma, Brazil) and dissolving it in 20 mL of 96° ethanol (Itajá, Jalles Machado, Brazil). For complete dissolution, this solution was placed in an ultrasonic bath at 40 kHz (Ultronique Q3.8/40A, Indaiatuba, São Paulo) for 3 min. From the stock solution, aqueous solution of 320 µM were prepared for use in the assays. Biofilm formation was determined by assessing the total biomass of yeasts adhered to the wells using the crystal violet method [ 33 ]. The Candida strains were stored under standard laboratory conditions (cryopreserved stocks at − 80 °C) and routinely maintained on Sabouraud agar prior to use. Yeasts were reactivated on Sabouraud agar (FIRSTLAB, MicroMedia, Hungary) incubated at 37 °C for 72 h. Subsequently, typical colonies were resuspended in 5 mL of sterile physiological saline (SPS), and the inoculum density was adjusted using the 0.5 McFarland scale. Then, 500 µL of the adjusted inocula were transferred to tubes containing 4,500 µL of Brain Heart Infusion broth with 2% sucrose (BHIS) (HIMEDIA ® , HiMedia Laboratories, India). After homogenization of the tubes, 100 µL of the broths with the inocula were transferred to wells of 96-well flat-bottom polystyrene microplates (Cralplast, Cral, Brazil), which were incubated at 37 °C for 48 h for biofilm formation. After incubation, the microplates were processed by removing the broth with total growth, washing the wells, fixing the adhered biofilms with 96% ethanol (Itajá, Jalles Machado, Brazil), staining with 0.1% crystal violet (Newprov, Brazil), removing excess dye with three washes with distilled water in an automatic microplate washer Aquari ® (MA 615, Brazil), and drying at room temperature (RT). Subsequently, 200 µL of 33% glacial acetic acid were added to the wells, and after incubation at RT for 20 min. Optical densities (OD 620nm ) were then read in a microplate spectrophotometer (MultiskanFC, ThermoScientific, China). Based on the OD 620nm values obtained from non-inoculated controls (NIC) and adhered yeasts (AY), biofilm formation was classified as weak (OD AY < 3xOD NIC ), moderate (3xOD NIC < OD AY 6xOD NIC ) [ 33 ]. The effect of curcumin-mediated photodynamic therapy on biofilm formation (preventive approach ) was evaluated as described for the biofilm formation study. Briefly, the microplate was prepared as previously mentioned with the addition of 100 µL of the adjusted inocula to wells in columns 2 to 11. In wells of columns 2 to 6, an additional 100 µL of the curcumin solution was added to achieve a final concentration of 320 µM. In the wells of the remaining columns, only the inoculum containing curcumin was added and maintained in the absence of light, serving as the non-photoactivated control (NPC). Yeasts were added to the microplate rows as follows: row B for C. albicans ATCC 10,231, row C for C. metapsilosis ATCC 96,143, row D for C. dubliniensis ATCC MYA-646, row E for C. orthopsilosis ATCC 96,141, row F for C. parapsilosis ATCC 22,019, and row G for the Non-Inoculated Control (NIC). Subsequently, the microplate was incubated in the absence of light at RT for 20 min (pre-irradiation time). Then, the microplate was illuminated for 1 h and 17 min (dose of 100 J/cm 2 ) using a 450 nm LED light source (0.02165 W/cm 2 ) to evaluate the effect of curcumin photoactivation on biofilm formation (prior to biomass establishment). Subsequently, the microplate was incubated at 37 °C for 48 h (Fig.  1 ). After the incubation period, the microplate was processed as previously described. Fig. 1 Evaluation of photodynamic therapy on biofilm formation and against pre-formed Candida spp. biofilms. 1. Incubation of Candida spp. on Sabouraud agar at 37°C for 72 hours 2. Adjustment of the inoculum density in Brain Heart Infusion broth with 2% sucrose (BHIS) to a 0.5 McFarland scale 3. Transfer of 100 μL of the adjusted inocula to microplate wells 4. Addition of 100 μL of 640 μM curcumin to the microplate wells 5. Incubation at RT for 20 minutes in the dark (pre-irradiation period) 6. Photoactivation with 450 nm LED light prior to biofilm formation 7. Incubation of the microplates at 37°C for 48 hours 8. Photoactivation with 450 nm LED light after biofilm formation 9. Measurement of OD 620nm and calculation of biofilm formation. OD 620nm  Optical density at 620 nm, CURC  curcumin, NPC  non-photoactivated control, NIC  non-inoculated control Evaluation of photodynamic therapy on biofilm formation and against pre-formed Candida spp. biofilms. 1. Incubation of Candida spp. on Sabouraud agar at 37°C for 72 hours 2. Adjustment of the inoculum density in Brain Heart Infusion broth with 2% sucrose (BHIS) to a 0.5 McFarland scale 3. Transfer of 100 μL of the adjusted inocula to microplate wells 4. Addition of 100 μL of 640 μM curcumin to the microplate wells 5. Incubation at RT for 20 minutes in the dark (pre-irradiation period) 6. Photoactivation with 450 nm LED light prior to biofilm formation 7. Incubation of the microplates at 37°C for 48 hours 8. Photoactivation with 450 nm LED light after biofilm formation 9. Measurement of OD 620nm and calculation of biofilm formation. OD 620nm  Optical density at 620 nm, CURC  curcumin, NPC  non-photoactivated control, NIC  non-inoculated control The activity of photodynamic therapy on mature biofilm was evaluated by exposing pre-formed biofilms under the same conditions described in item 2.3. Microplates were prepared as previously mentioned for biofilm formation assays with incubation at 37 °C for 48 h. After mature biofilms were formed, the microplates were treated under aseptic conditions by removing the spent media and adding 100 µL of the 320 µM curcumin solution to the wells, as described in item 2.4. Subsequently, the microplates were incubated for 20 min (pre-irradiation time) at RT in the dark. After the pre-irradiation period, the microplate was illuminated for 1 h and 17 min (dose of 100 J/cm 2 ) using a 450 nm LED light source (0.02165 W/cm 2 ) to evaluate the impact of curcumin-mediated photoactivation against mature (48 h) biofilm (Fig.  1 ). After the incubation period, the microplate was processed as previously described in item 2.3. All experiments were performed in independent triplicates, and the results are presented as mean ± standard deviation. The OD 620 nm values obtained from photoactivated wells containing curcumin were compared to those from non-photoactivated control wells (curcumin without light exposure). Data distribution was assessed using the Shapiro-Wilk normality test. For the biofilm formation assay, normality was not observed ( p  = 0.007), and data were therefore analyzed using the Kruskal-Wallis test followed by Dunn’s post hoc test for multiple comparisons. In this essay, curcumin-treated groups were compared with their respective untreated controls, with statistical significance defined as p  < 0.05. For the mature biofilm assay, the Shapiro–Wilk test did not reject normality (p  = 0.284). Nevertheless, considering the small sample size and maintaining a conservative analytical framework across assays, non-parametric analysis was retained. Comparisons were performed using the Kruskal-Wallis test. No statistically significant differences were detected between curcumin-treated mature biofilms and their respective untreated controls. Statistical analyses were performed using Jamovi and BioEstat. Data are presented as mean ± standard deviation.

Biofilms are complex, three-dimensional microbial communities comprising single or multiple species. They can attach to biological surfaces, like host tissues, or abiotic surfaces, such as medical devices. These communities are enveloped in an extracellular polymeric substance (EPS) matrix, primarily composed of polysaccharides, proteins, and nucleic acids. This matrix acts as a protective barrier, making biofilms resistant to antimicrobial agents and host immune responses [ 21 ]. Infections caused by human pathogenic fungi, particularly Candida , represent a significant medical challenge due to their ability to disseminate throughout the body. Candida infections associated with biofilms are especially difficult to treat as they are recalcitrant and exhibit resistance to antifungals. The limited availability of therapeutic classes and antifungals further complicates treatment [ 16 ]. Research on C. albicans biofilms indicates that preventing their initial formation is more effective than treating mature, more resistant biofilms [ 5 ]. One alternative strategy to inhibit C. albicans biofilm formation involves modifying the chemical properties of biomaterial surfaces to prevent or reduce biofilm development [ 13 ]. Alternative therapies are being explored to combat fungal biofilm infections. These include phytoextracts and nanoparticles, which can act as carriers for EPS matrix disrupters [ 1 ]. Antimicrobial photodynamic chemotherapy (aPDT) is another promising approach, involving the use of a light source, molecular oxygen (O 2 ) and a photosensitizer (PS) to generate reactive oxygen species (ROS), leading to cell death. This therapy is being investigated against various microbial biofilms [ 13 ]. Photosensitizers, such as tetrapyrroles, synthetic dyes, and natural compounds, are activated by specific light wavelengths in the presence of oxygen to produce ROS. This ROS production induces oxidative stress and cytotoxicity within the cell, resulting in microorganism death [ 31 ]. Among the PS applied, curcumin is able to generate ROS that cause severe and irreversible oxidative stress in microbial cells. This multifaceted attack targets various cellular components simultaneously. For instance, the plasma membrane undergoes lipid peroxidation, leading to structural integrity loss, increased permeability, and the leakage of intracellular contents. ROS also oxidizes and denatures essential proteins and enzymes. Furthermore, oxidative damage to nucleic acids (DNA and RNA) can cause strand breaks and mutations, rendering genetic replication and transcription inviable [ 12 ]. The broad range of mechanisms and low propensity for resistance selection associated with photodynamic inactivation of microorganisms lead to a combination of cellular structure and function damage, resulting in rapid death of microorganisms, including Gram-positive and Gram-negative bacteria, fungi, and enveloped viruses [ 9 , 10 , 24 ]. Given the increasing resistance of yeasts to conventional antifungals and the challenges in managing Candida biofilm-associated infections [ 13 , 25 ], this study aimed to evaluate the efficacy of curcumin-mediated aPDT using 450 nm LED light in both inhibiting biofilm formation (preventive approach) and reducing pre-formed mature biofilms of five standard Candida strains.