Dental caries is the most frequent biofilm-related human infectious disease in the oral cavity. Streptococcus mutans is one of the primary etiological agents of dental caries. The aim of our study was to investigate the effects of rhein-8-O-β-D-glucopyranoside (Rg) on the development of S. mutans biofilms. Growth curves were generated, and biofilm oxygen sensitivity was detected after Rg treatment. The expression levels of luxS, brpA, ffh, recA, nth, and smx were analyzed by real-time PCR. The trypan blue exclusion assay was used to measure the effect of Rg on monocyte viability. The results showed that Rg could significantly inhibit the growth of S. mutans and suppress the biofilm formation of S. mutans in a concentration-dependent manner. In Rg-treated biofilms, the expression levels of luxS, brpA, ffh, recA, nth, and smx were all decreased. Our results further showed that Rg was nontoxic, as Rg did not affect monocyte viability or lactate dehydrogenase activity in the exposed cells. These results suggested that Rg inhibited the biofilm formation of S. mutans, and the decrease in luxS, brpA, ffh, recA, nth, and smx expression might contribute to the antibacterial effects of Rg.