Objectives: The objectives of the study were to evaluate and compare the antimicrobial property of nanochitosan coated and uncoated stainless steel (SS) brackets against Streptococcus mutans and Lactobacillus acidophilus. Materials and Methods: 22 SS orthodontic brackets coated with nanochitosan and 22 uncoated brackets were bonded to the crowns of extracted premolar teeth and prepared for the antimicrobial assay against S. mutans and L. acidophilus. The bacterial suspensions were incubated in Mueller Hinton broth and cultivated in Petri dish plates. The bacterial colonies were counted after 1, 6, 12, 24, and 72 h intervals using a digital colony counter. Inter and intragroup comparisons were done using independent sample t-test and repeated measures ANOVA (P ≤ 0.05). Results: The mean colony-forming units (CFU) of S. mutans and L. acidophilus showed a significant reduction in the coated brackets from 1 h to 72 h (P = 0.000). The mean CFU of S. mutans and L. acidophilus showed a significant increase in the uncoated brackets from 1 h to 24 h and decreased thereafter at 72 h (P = 0.000) except for S. mutans between 12 h and 72 h where the difference was not statistically significant (P = 0.837). The Mean CFU of S. mutans and L. acidophilus was significantly lesser in coated brackets compared to uncoated brackets at all-time intervals (P = 0.000). Conclusion: Nanochitosan coated orthodontic brackets showed significant antimicrobial properties against S. mutans and L. acidophilus in short-term up to 72 h compared to uncoated SS brackets.