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Statement of problem Clinical evidence on the best chemical protocol for the disinfection and removal of biofilm from complete dentures is lacking. Purpose The purpose of this crossover randomized clinical trial was to assess the effectiveness of various chemical hygiene clinical protocols in reducing the microbial viability of biofilm formed on complete dentures. Material and methods In this triple-blind (participants, dentist, and outcome evaluator) study, complete denture wearers without candidiasis were randomly divided into 4 groups (n=40) according to the chemical hygiene protocol: water (placebo), 0.5% sodium hypochlorite solution, 0.12% chlorhexidine gluconate solution, and 5% sodium bicarbonate solution. The biofilm formed on the palate intaglio and denture teeth was collected and assessed in each experimental phase for quantitative microbial viability at the seventh and 14th day after using the chemical protocol. Results Two participants were lost. Data were analyzed by MANOVA and Tukey HSD tests. Soaking dentures was not effective in decreasing Candida albicans, C. non-albicans, and lactobacillus counts. The use of sodium hypochlorite and chlorhexidine decreased total microorganisms and Streptococcus mutans counts for both palate and teeth compared with water and sodium bicarbonate. The intaglio of the dentures always presented higher microbial counts than did the denture teeth. Conclusions The use of sodium hypochlorite and chlorhexidine and mechanical cleansing with a toothbrush decreased microbial viability in healthy complete denture wearers.