Objective Currently, dental implants are a predictable treatment option for oral rehabilitation; however, complications such as peri-implant diseases are increasing every day. Thus, the aim of this study was to verify the efficacy, in vitro, of two protocols against cultures of periodontal biofilm and Staphylococcus aureus. Material and Methods Petri dishes for each of the following groups were used: control groups (C)—plates inoculated with periodontal biofilm (C.B; n = 4) or S. aureus (C.SA; n = 4) without any treatment; laser groups—plates inoculated with periodontal biofilm (low-level laser therapy [LLLT].B; n = 4) or S. aureus (LLLT.SA; n = 4) and treated with LLLT (660 nm, 30 mW, 50 J/cm2, and 47 seconds); antimicrobial photodynamic therapy groups (aPDT)—plates inoculated with periodontal biofilm (aPDT.B; n = 4) or S. aureus (aPDT.SA; n = 4) and treated with aPDT (red laser 660 nm, 30 mW, 50 J/cm2, 47 seconds + toluidine blue O (TBO) 100 µg/mL, and 1 minute). After treatments were performed, the contents of all plates were diluted and seeded for counting colony-forming units (CFUs). Statistical Analysis Results were analyzed with one-way analysis of variance (ANOVA), Tukey’s test, comparison of percentages, and independent t-tests with a 5% significance level. Results Both treatments, LLLT and aPDT, significantly reduced the number of CFUs for the two types of culture, LLLT.B (3.69 × 106 ± 0.20), aPDT.B (2.79 × 106 ± 0.13), LLLT.SA (4.10 × 106 ± 0.12), and aPDT.SA (3.23 × 106 ± 0.10) when compared with control groups C.B (5.18 × 106 ± 0.43) and C.SA (5.81 × 106 ± 0.16; p = 0.000). When treatment groups were compared separately, there was also a statistically significant difference (p = 0.000). None of the protocols were able to eliminate cultured microorganisms. Conclusion The LLLT and aPDT protocols effectively reduced cultures of periodontal biofilm and S. aureus in vitro, with the superiority of aPDT.
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