BACKGROUND : Soaking dentures in vinegar or hydrogen peroxide does not seem to remove the microorganisms involved with prosthetic stomatitis efficiently. A mixture of these 2 substances may be effective, but studies are lacking. OBJECTIVE : The purpose of this in vitro study was to evaluate the antimicrobial effect and cytotoxic activity of vinegar-hydrogen peroxide mixtures against Candida albicans and Staphylococcus aureus. METHODS : For antimicrobial tests, planktonic cells and biofilms of C. albicans and S. aureus cultured on acrylic resin disks were exposed to 0.5% sodium hypochlorite; 0.2% peracetic acid; vinegar-hydrogen peroxide mixtures at concentration ratios 1:1, 1:3, and 3:1; vinegar-water mixtures at concentration ratios 1:1, 1:3, and 3:1; and hydrogen peroxide-water mixtures at concentration ratios 1:1, 1:3, and 3:1. Antimicrobial activity was evaluated by counting viable colony-forming units after disinfection. For cytotoxicity tests, the 1:1 vinegar-hydrogen peroxide mixture was serially diluted (10-1 to 10-4) and allowed to be in direct contact with HaCaT keratinocytes for 24 hours. Cytotoxicity was quantitatively and qualitatively determined by counting the number of viable cells and analyzing morphological cell changes. RESULTS : All vinegar-hydrogen peroxide mixtures, sodium hypochlorite, and peracetic acid efficiently eliminated C. albicans and S. aureus (P<.05), whereas vinegar and hydrogen peroxide solutions used separately were not as efficient as the experimental mixtures. The 10-3 and 10-4 dilutions of vinegar-hydrogen peroxide solutions were considered noncytotoxic, whereas dilutions below 10-2 were strongly cytotoxic, comparable with the 10-2 dilution of 0.2% peracetic acid. CONCLUSIONS : The vinegar-hydrogen peroxide mixture effectively eliminated C. albicans and S. aureus from acrylic resin. Dilutions equal or below 10-2 of this mixture presented strong cytotoxic effects.