Background: Oral microbiota comprises a wide variety of microorganisms. The purpose of this study was to evaluate the effects of Nd:YAG laser with a 1064 nm wavelength on the in vitro growth of Candida albicans, Candida glabrata, and Streptococcus mutans clinical strains, as well as their biofilm. The study also aimed to determine whether the parameters recommended for photobiomodulation (PBM) therapy, typically used for tissue wound healing, have any additional antibacterial or antifungal effects. Material and Methods: Single- and dual-species planktonic cell solution and biofilm cultures of Streptococcus mutans, Candida albicans, and Candida glabrata were irradiated using an Nd:YAG laser (LightWalker; Fotona; Slovenia) with a flat-top Genova handpiece. Two test groups were evaluated: Group 1 (G-T1) exposed to low power associated parameters (irradiance 0.5 W/cm²) and Group 2 (G-T2) with higher laser parameters (irradiance 1.75 W/cm²). Group 3 (control) was not exposed to any irradiation. The lasers’ effect was assessed both immediately after irradiation (DLI; Direct Laser Irradiation) and 24 h post-irradiation (24hLI) of the planktonic suspension using a quantitative method (colony-forming units per 1 mL of suspension; CFU/mL), and the results were compared with the control group, in which no laser was applied. The impact of laser irradiation on biofilm biomass was assessed immediately after laser irradiation using the crystal violet method. Results: Nd:YAG laser irradiation with photobiomodulation setting demonstrated an antimicrobial effect with the greatest immediate reduction observed in S. mutans, achieving up to 85.4% reduction at the T2 settings. However, the laser’s effectiveness diminished after 24 h. In single biofilm cultures, the highest reductions were noted for C. albicans and S. mutans at the T2 settings, with C. albicans achieving a 92.6 ± 3.3% reduction and S. mutans reaching a 94.3 ± 5.0% reduction. Overall, the T2 settings resulted in greater microbial reductions compared to T1, particularly in biofilm cultures, although the effectiveness varied depending on the microorganism and culture type. Laser irradiation, assessed immediately after using the crystal violet method, showed the strongest biofilm reduction for Streptococcus mutans in the T2 settings for both single-species and dual-species biofilms, with higher reductions observed in all the microbial samples at the T2 laser parameters (p < 0.05) Conclusion: The Nd:YAG laser using standard parameters typically applied for wound healing and analgesic effects significantly reduced the number of Candida albicans; Candida glabrata; and Streptococcus mutans strains.

Purpose: This in vitro study aimed to evaluate the antimicrobial effects of a 1064 nm Nd:YAG laser delivered through a novel handpiece designed to provide a flat-top, homogeneous energy distribution profile (MarcoM handpiece) against key oral pathogens: Candida albicans, Candida glabrata, and Streptococcus mutans, in both planktonic and biofilm forms, in single- and dual-species cultures. Methods: Microorganisms were cultured individually and in dual-species combinations and irradiated using two irradiance settings (T1: 0.2 W/cm², T2: 0.6 W/cm²) corresponding to clinically established photobiomodulation parameters for wound healing and analgesia. Colony-forming units (CFU/mL) were quantified immediately and 24 hours post-irradiation to assess laser-induced antimicrobial effects.Results: Streptococcus mutans exhibited the highest susceptibility in planktonic form, with CFU reductions of 79.0 ± 5.6% (T1) and 86.3 ± 4.2% (T2) immediately after irradiation (p < 0.05). After 24 hours, a 92.0 ± 3.5% reduction was observed, though viability partially recovered in dual-species cultures with C. albicans (T1: 4.7 ± 6.6%, T2: 0.0 ± 0.0%). C. albicans showed greater reduction after 24 hours (T2: 68.2 ± 9.7%) than immediately post-irradiation (32.1 ± 6.7%) (p < 0.05). In biofilm forms, the greatest effect was observed for C. albicans at T2 (91.7 ± 2.8%), while C. glabrata biofilms showed no significant difference between settings (p > 0.05). Dual-species biofilms required higher irradiance (T2, p < 0.05) for significant eradication.Conclusion: Under the specific in vitro conditions tested, 1064 nm Nd:YAG irradiation delivered with a flat-top, homogeneous beam profile reduced CFU counts and biofilm biomass of the tested oral microbial isolates. The effect depended on laser parameters, microbial species, culture model, and time point. Because only one clinical isolate per species was used and no positive antimicrobial comparator was included, these findings should be interpreted as preliminary proof-of-concept data requiring validation in expanded strain panels and comparator-controlled studies.