Streptococcus mutans (S. mutans) is a bacterium that colonizes in the mouth and is a common cause of dental caries and periodontal diseases. This bacterium comprises 70% of the bacteria in the dental plaque. Although tooth decay is a multifactorial complication, S. mutans biofilms are the main cause of cavitated carious lesions. Considering the importance of this microorganism, we aimed at investigating the effect of photodynamic therapy (PDT) using curcumin (CUR) and methylene blue (MB) photosensitizers on S. mutans. In this in-vitro experimental study, first, samples of S. mutans were prepared in 110 test tubes and were randomly assigned to 11 groups after colony counting: 1) Positive control group, 2) Negative control group, 3) CUR extract group, 4) 460-nm laser group, 5) 460-nm continuous laser + CUR group, 6) 460-nm discontinues 50% duty cycle (DC) laser + CUR group, 7) 660-nm laser group, 8) 660-nm laser + MB group, 9) MB group, 10) dental light-curing group, and 11) chlorhexidine (CHX) group. After the intervention, cultivation was performed again in blood agar medium, and the bacterial colony-forming units per milliliter (CFU/ml) were counted again. Data were analyzed using analysis of variance (ANOVA) and Tukey's test. CHX and 460-nm low-level continuous laser + CUR had the highest and most significant effect on inhibiting the growth of S. mutans bacterial colonies and showed significant differences with other groups (P < 0.001). According to the results, MB- and CUR-mediated PDT can significantly eradicate S. mutans colonies.