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Purpose: To evaluate the application of chitosan as a cleanser in the control of biofilm formation on cobalt-chromium (Co-Cr) alloy and acrylic resin surfaces.Materials and Methods: In total, 172 Co-Cr discs and 172 acrylic resin discs (14 mm x 3 mm) were contaminated with Streptococcus mutans, Staphylococcus aureus, Candida albicans, or Candida glabrata and incubated for 48 hours. Then, specimens were randomly divided into groups and immersed in the following solutions for 15 minutes: WC = solution without chitosan (control); CH = chitosan solution (5 mg/mL); CN = chitosan nanoparticle solution (3.8 mg/mL); and ET = effervescent tablet. Biofilm recovery rates (n = 9) were evaluated by counting the colony-forming units (CFU/mL). Biofilm morphology was evaluated using scanning electron microscopy. Data were compared with Kruskal-Wallis or analysis of variance followed by Tukey post hoc tests.Results: For acrylic resin, ET showed the lowest number of CFU for S aureus and S mutans (P < .001). CH exhibited intermediate values for S mutans, S aureus, and C albicans, and CN exhibited intermediate values for S mutans and S aureus. For C glabrata, there was no statistical difference between the solutions (P = .264). For Co-Cr, ET showed the highest level of antimicrobial action against all microorganisms (P < .001), and CH showed an intermediate level of action against S mutans and S aureus. Against C albicans and C glabrata, there was no significant difference among CH, CN, and WC.Conclusion: Although ET had a broader spectrum of antimicrobial action, the chitosan solution showed promise as a denture cleanser. Int J Prosthodont 2021.