Objective: Polyhexanide (PHMB; polyhexamethylene biguanide) is a well-known antiseptic agent; however, no data exist for its applicationon denture base and lining materials. In the present in vitro study, the aim was to compare viable bacterial or fungal cell numbers and theiradhesion on different types of denture base and lining materials in diverse concentrations of PHMB.Methods: Light-activated urethane dimethacrylate (UDMA), heat-polymerized polymethyl methacrylate (PMMA), heat-polymerizedpolydimethylsiloxane, and autopolymerizing polyethylmethacrylate discs were prepared for each group (n = 10). 1×108 CFU/mL of all the testedspecies were appended separately to discs, and they were immersed into different PHMB suspensions (0.5%, 1%, 2%, and 5%) for 10 minutes.The antimicrobial activity and number of adherent species on the surface were evaluated.Results: In the PMMA group, all studied species except C. albicans, L. acidophilus, and S. aureus were decreased in various concentrations (p <0.05), and all studied species presented a significant decrease in every concentration of PHMB in the UDMA group (p < 0.01) in comparison to thecontrol. N. sicca, K. pneumoniae, S. pyogenes, S. sanguis, C. pseudotuberculosis, and S. aureus (p < 0.05) were reduced in the heat-polymerizedpolydimethylsiloxane group, while all tested species except B. subtilis were decreased in the autopolymerizing polyethylmethacrylate group incomparison to the control (p < 0.01). Among all tested materials and species, no significant difference was detected in adherent cell number(p > 0.05).Conclusion: PHMB suspension, in various concentrations, can reduce some species of bacterial and yeast cells.