As denture cleansers should be able to reduce biofilm accumulation without changing the acrylic resin surface structure, this study evaluated the effect of denture cleansers on surface roughness of acrylic resin and on biofilm accumulation. It was conducted a crossover study of three phases of 4 days each and 13 volunteers wore palatal appliances containing four specimens of acrylic resin of known surface roughness which were extra-orally submitted once a day to three groups of separated treatments: (i) negative control, (ii) enzymatic commercial solution (Ortoform) or (iii) 0·5% sodium hypochlorite (NaOCl).The appliances were immersed eight times a day in a 20% sucrose solution to enhance biofilm formation. On morning of the fifth day of each phase, the amount of biofilm formed on the specimens was estimated by the amount of protein extracted with alkali and the surface roughness of the specimens was again measured. New palatal appliances with new specimens were worn by the volunteers in the following phases, but the treatments were changed. The roughness of the resin increased after the treatments (P < 0·05) but the difference among the cleansers was not statistically significant (P = 0·85). The lowest amount of biofilm formed on acrylic resin specimens was found for the treatment with NaOCl (P < 0·05) but the enzymatic product did not differ from the negative control group (P > 0·05). The data suggest that the roughness of acrylic resin was not changed by the cleansers, but the ability to reduce biofilm accumulation depended on the product used.