Background: Tempeh, the Southeast Asian traditional food, has garnered great attention for its antibacterial property against Gram-positive and Gram-negative pathogens, and antidiarrheal effect. We have previously reported the potentiality of tempeh hexane fraction (HXF) in ceasing Actinomyces viscosus biofilm formation in vitro. Objective: Here, we investigated the efficacy of tempeh HXF on other cariogenic virulence traits of A. viscosus such as adhesive properties, acid production, and plaque growth. Materials and Methods: Anti adhesion of HXF was assessed based on its effects on the number of cells adhering to the surface of tooth in sucrose-dependent (SD) and sucrose-independent (SI) medium. The potential of HXF to inhibit the capability of A. viscosus to generate acids was investigated by pH drop assay. The HXF at different concentrations were used to determine the LC50based on brine shrimp lethality assay. Finally, the prospect of HXF as an inhibitor of plaque formation was investigated using artificial saliva-coated denture as an in vitro batch model. Results: HXF significantly decreased colony-forming unit of SD (1.07 log reduction) and SI (0.56 log reduction)-mediated adsorption of bacterial cells onto the tooth surface over 4- and 12-h incubation, respectively. Acid production was reduced after treated with HXF in a dose-dependent manner. Finally, a substantial reduction in plaque coverage area >55% was found on the HXF treated-denture. Conclusion: The anti-biofilm effect of HXF was associated with the suppression of A. viscosus adhesion to tooth surfaces and reduction in acid production. Furthermore, in vitro anti-plaque potential of HXF was demonstrated.