Mushrooms contain large quantities of alpha-glucans. Shiitake (Lentinula edodes), Japan's most popular edible mushroom, has been reported to contain about 6% (weight/dried weight) of alpha-(1,3)-glucan. This glucan is one of the major components of oral biofilm formed by the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. We found that extracts from shiitake and other edible mushrooms could reduce preformed biofilms of S. mutans and S. sobrinus in the presence of dextranase. We also investigated the alpha-glucanase activities of shiitake mushroom extracts and their effects on biofilm formation. The extracts possessed alpha-glucanase activity and degraded water-insoluble glucans from mutans streptococci. The extracts strongly inhibited the sucrose-dependent formation of biofilms by S. mutans and S. sobrinus in the presence of dextranase. Our results suggest that some components of mushrooms, including alpha-glucanases, might inhibit the sucrose-induced formation of oral biofilms.