The production of β-1,3-glucanase has been optimized using vinasse, analyzing the ideal concentration, incubation time, pH, temperature, and agitation. The biofilms were formed on stainless steel coupons for 48 hours and treated with 21.3 U/mg of enzyme for 30 minutes. The degradation efficiency of the β-1,3-glucanase produced was evaluated by the reduction of total biomass, using crystal violet staining. The enzyme showed significant biomass degradations of microbial biofilms and had the best action against Candida albicans biofilms, with total biomass reduction of 48.76%.These results are important to provide a practicable alternative for elimination of biofilms, employing agro-industrial residues that accumulate in environment.