ObjectiveTo evaluate the anti‐biofilm action of chitosan, nanoparticulate chitosan, and denture cleanser Nitradine™ against biofilms comprising Candida albicans, Candida glabrata, Staphylococcus aureus, and Streptococcus mutans.BackgroundBiofilm removal from removable partial dentures (RPD) is important for success in prosthetic rehabilitation.Materials and MethodsThe anti‐biofilm action of the experimental chitosan‐based solutions and Nitradine™ was evaluated on acrylic resin and cobalt‐chromium alloy through assessing cell viability, cell metabolism, residual aggregated biofilm, and extracellular polymeric substance and biofilm morphology.ResultsOnly chitosan reduced the viability of C. albicans on cobalt‐chromium alloy surface, by 98% (a 1.7 log10 reduction in cfu). Chitosan‐based solutions neither promoted substantial alteration of the metabolic activity of the four‐species biofilm nor reduced the amount of the aggregated biofilm. After immersion in chitosan and nanoparticulate chitosan, viable microorganisms and extracellular polymeric substances distributed over the entire specimens' surfaces were observed. Nitradine™ reduced the viability and metabolic activity of biofilm grown on both surfaces, but it did not remove all aggregated biofilm and extracellular polymeric substances. After immersion in Nitradine™, approximately 35% of the specimens' surfaces remained covered by aggregated biofilm, mainly composed of dead cells.ConclusionAlthough chitosan and Nitradine™ promoted changes in the viability of microorganisms, neither solution completely removed the four‐species biofilm from the Co–Cr and acrylic resin surfaces. Thus, isolated use of hygiene solutions is not indicated for biofilm control on RPDs; this requires complementary mechanical removal.