Background: Pit and fissure sealant therapy is widely used to prevent caries of the occlusal surfaces of posterior teeth. In this treatment, failure is not uncommon. Streptococcus mutans is the most important bacterium that initiates and progresses caries lesions. This study aimed to evaluate the synergizing antibacterial effect of chlorhexidine solution 0.2% and phosphoric acid gel 37% against S. mutans during fissure sealant therapy to reduce the risk of failure. The disc diffusion method was used to evaluate the effects of the materials. This method has been applied in many studies. Methods: The antibacterial effect of the two materials was evaluated using the disc diffusion method. S. mutans was acquired from the Iranian Industrial Bacteria and Fungi Collection Center (code ATCC35668) and was cultured in a laboratory environment on three plates containing a blood agar culture medium. In each plate were placed four discs: a disc that had been soaked in 0.2% chlorhexidine solution, a disc that had been soaked in 37% phosphoric acid, a disc that had been soaked in sterile distilled water as a negative control, and a disc containing ciprofloxacin antibiotic as a positive control. The plates were placed in the incubator for specific periods. The diameter of the bacteria inhibition halo around the test and control discs was measured and compared with each other. SPSS 26 statistical software was used for data analysis. The Mann-Whitney test was performed, and descriptive statistics were used to describe the halo of the bacterial inhibition growth. Results: Phosphoric acid gel 37% showed significant anti-S. mutans effect. The antibacterial effect of chlorhexidine 0.2% solution was also comparable to that of the positive control disc. Conclusion: Applying chlorhexidine solution on the occlusal surfaces shortly before the sealant therapy can reduce the risk of decay under the sealants.