Allyl isothiocyanate (AITC) is a naturally occurring, pungent compound abundant in cruciferous vegetables and functions as a repellent for various organisms. The antibacterial effect of AITC against various bacteria has been reported, but there are no reports on the effect on , a major bacterium contributing to dental caries. In this study, we investigated the inhibitory effect and mechanism of AITC on the survival and growth of . AITC showed an antibacterial effect in a time- and concentration-dependent manner. In addition, bacterial growth was delayed in the presence of AITC, and there were almost no bacteria in the presence of 0.1% AITC. In a biofilm assay, the amount of biofilm formation with 0.1% AITC was significantly decreased compared to the control. RNA sequencing analysis showed that the expression of 39 genes (27 up-regulation and 12 down-regulation) and 38 genes (24 up-regulation and 14 down-regulation) of was changed during the survival and the growth, respectively, in the presence of AITC compared with the absence of AITC. Protein-protein interaction analysis revealed that AITC mainly interacted with genes of unknown function in . These results suggest that AITC may inhibit cariogenicity of through a novel mechanism.