Introduction: This study aimed to assess the effect of photodynamic therapy (PDT) and chitosan separately and in combination on Streptococcus mutans. Methods: This in vitro experimental study evaluated 216 microbial samples in 6 groups. First, 5 µL of 0.5 McFarland standard suspension of S. mutans was added to each well of an ELISA microplate; 100 µL of Mueller Hinton broth was also added to each well; 180 wells contained S. mutans suspension while 36 wells were devoid of bacteria. Group 1 served as the negative control and had no bacteria. Group 2 served as the positive control and S. mutans in the positive control wells did not undergo any intervention. In groups 3 and 4, PDT with a 50 mW low-level laser was performed for 30 and 40 seconds respectively. In group 5, 3 mg/mL of chitosan (100 µL) was used. In group 6, 3 mg/mL (100 µL) of chitosan was used in combination with PDT (50 mW laser for 30 seconds). The laser was irradiated under aseptic conditions at a 660 nm wavelength with 50 mW power. Data were analyzed using one-way ANOVA and Tukey's test. Results: PDT combined with chitosan showed maximum bactericidal effect followed by PDT for 40 seconds and chitosan groups (P < 0.05). PDT for 30 seconds showed a minimum bactericidal effect (P < 0.05). All pairwise comparisons revealed significant differences (P < 0.001). Conclusion:Chitosan and PDT alone can be used to decrease the S. mutans count. However, their combined use has a greater bactericidal effect on S. mutans.