Objective: To investigate the antimicrobial activity of colloidal selenium nanoparticles in chitosan solution (Cts-Se-NPs) against Streptococcus mutans, Lactobacillus acidophilus, and Candida albicans. Material and Methods: Cts-Se-NPs solution was prepared using a simple chemical reduction method. The MIC and MBC against S. mutans, L. acidophilus, and C. albicans were determined using the broth dilution assay. Results: The Cts-Se-NPs had remarkable antimicrobial activity against S. mutans, L. acidophilus, and C. albicans. The MIC values of the Cts-Se-NPs were lowest for S. mutans (0.068 mg/ml) compared to L. acidophilus (0.137 mg/ml), and C. albicans (0.274 mg/ml). The MBC values of the Cts-Se-NPs against the microorganisms after one, two, six, and 24 hours indicated that the concentration of 0.274 mg/ml of Cts-Se-NPs completely killed S. mutans, L. acidophilus, and C. albicans after one, two, and six hours, respectively. At the concentration of 0.137 mg/ml, S. mutans and L. acidophilus were killed after six and 24 hours, respectively. Conclusion: These findings encourage the potential use of Cts-Se-NPs in dentistry, while further clinical research is required in this area.