Introduction: Streptococcus viridans is the most common facultative anaerobic bacteria which can cause pulp and root canal infections in oral cavity. This Gram-positive bacterium can cause pulp necrosis, an indication for root canal treatment. One of the important steps in root canal treatment is root canal irrigation and a good root canal irrigant has a maximum antibacterial effect with minimum toxicity effect. Chitosan is a natural ingredient from shrimp shells. Chitosan has high antibacterial activity against many pathogenic microorganisms and has been used as an antibacterial agent to eliminate Streptococcus viridans. Methods: Streptococcus viridans was grown in a tube which contained BHIB medium and 2% chitosan suspension in each sample group with the concentration of 0.0156%; 0.03125%; .0625%; 0.125%; 0.25%; 0.5%; 1%; 2%. Positive control (BHIB medium and Streptococcus viridans bacteria), 2% acetic acid solution control (2% acetic acid solution and Streptococcus viridans), and negative control (BHIB medium) were made to ensure that there was not any antibacterial effect on the medium. Each tube was incubated at 37°C for 24 hours. The number of living colonies were calculated and compared with the positive control to determine the antibacterial activity, MIC, and MBC. Results: In this study, the percentage of the living colonies in the sample groups with chitosan concentration of 0.125%; 0.0625%; 0.03125% were 0%; 7.2%; 16.4%, respectively. Conclusion: Chitosan has an antibacterial activity with 0.0625% MIC and 0.0125% MBC against Streptococcus viridans.