Polygonum cuspidatum is a plant with spreading rhizomes and numerous reddish-brown stems that has been used in Korean folk medicine to improve oral hygiene. Nevertheless, there are no reports related to its possible effect on the virulence of dental biofilms. In this study, the ability of a fraction (F1) separated from P. cuspidatum, alone or in combination with fluoride, to disrupt virulence factors and the composition of Streptococcus mutans biofilms was examined. F1 was mainly composed of resveratrol, emodin and physcion (approximately 16.2%, 18.9% and 2.07% of the weight of F1, respectively). F1 showed inhibitory effects on acid production and F-ATPase activity of S. mutans in biofilms, and could enhance fluoride activity against acid production and acid tolerance of S. mutans in biofilms. When S. mutans biofilms were briefly treated with F1 (10 min, a total of five times), the biomass accumulation, water-insoluble polysaccharides and intracellular iodophilic polysaccharides were reduced. Furthermore, the fluoride activity against biomass accumulation was enhanced by F1. These results suggest that F1 may be useful in the control of dental biofilms and in improving the cariostatic properties of fluoride without increasing its exposure.