Abstract: Streptococcus mutans is a primary etiological agent of dental caries. Farnesol, as a potential antimicrobial agent, inhibits the development of S. mutans biofilm. In this study, we hypothesized that farnesol inhibits caries development in vitro and interferes with biofilm formation by regulating virulence-associated gene expression. The inhibitory effects of farnesol to S. mutans biofilms on enamel surfaces were investigated by determining micro-hardness and calcium measurements. Additionally, the morphological changes of S. mutans biofilms were compared using field emission scanning electron microscopy and confocal laser scanning microscopy, and the vitality and oxygen sensitivity of S. mutans biofilms were compared using MTT assays. To investigate the molecular mechanisms of farnesol’s effects, expressions of possible target genes luxS, brpA, ffh, recA, nth, and smx were analyzed using reverse-transcription polymerase chain reaction (PCR) and quantitative PCR. Farnesol-treated groups exhibited significantly higher micro-hardness on the enamel surface and lower calcium concentration of the supernatants as compared to the-untreated control. Microscopy revealed that a thinner film with less extracellular matrix formed in the farnesol-treated groups. As compared to the-untreated control, farnesol inhibited biofilm formation by 26.4% with 500 μmol/L and by 37.1% with 1,000 μmol/L (P < 0.05). Last, decreased transcription levels of luxS, brpA, ffh, recA, nth, and smx genes were expressed in farnesol-treated biofilms. In vitro farnesol inhibits caries development and S. mutans biofilm formation. The regulation of luxS, brpA, ffh, recA, nth, and smx genes may contribute to the inhibitory effects of farnesol.