Background: The current procedures for treating tooth decay are not always guaranteed to successfully remove all microorganisms which cause disease. For elimination of bacteria and prevention of their effects, different methods are recommended, such as antibacterial materials and cavity disinfectants. The aim of this study was to compare the antibacterial activity of photodynamic therapy (PDT) using diode laser with indocyanin green (ICG) on Streptococcus (S.) mutans biofilm with conventional methods. Methods: Ninety human molars were divided into 9 groups: negative control, positive control, CHX, NaOCl, gaseous ozone, erbium (Er):YAG laser, diode laser, and indocyanin green/ICG, and PDT. Cavities were then cut into the teeth (2 per tooth, 20 cavities per group) and sterilized. For all of the groups, with the exception of the negative control group, 10 5 CFU/mL of the active S. mutans culture were inoculated into the cavities and then incubated at 37 °C for 72 h. Then, dentin chips (25 ± 5 mg) were collected from cavity walls and spread on Mueller-Hinton agar media after decimal dilution. The colonies were counted after incubation at 37 °C for 24 h. Results: All the treatments significantly reduced the number of S. mutans compared with the positive control group (p < 0.05). The antimicrobial effectiveness of CHX, NaOCl, ozone, PDT, Er:YAG laser, and diode laser groups were similar. The lowest bacterial reduction was observed in the ICGgroup. Conclusions: This work concludes that PDT using diode laser with ICG may be suggested on the cavity disinfection after caries excavation as an alternative to conventional methods.