Vine tea (Ampelopsis grossedentata) has been approved as a new food ingredient in 2013. Both vine tea extract (VTE) and its active ingredient, 2R, 3R-Dihydromyricetin (DMY), showed good antibacterial activity. The mechanism of VTE and DMY against Staphylococcus aureus were evaluated by morphology observation, cell membrane and wall assay, protein assay, and DNA assay in this study. The results of SEM and TEM revealed that the VTE and DMY changed the morphology of S. aureus. The leakage of AKPase and β-galactosidase in treated groups demonstrated that the membrane integrity of S. aureus was disrupted. Meanwhile, the results of protein assay showed that VTE and DMY inhibited the expression of total proteins, and decreased activities of a few energy metabolism enzymes, total ATPase. Moreover, spectral and competitive analysis revealed that VTE and DMY interacted with DNA by groove and intercalation binding. Finally, the suspension experiments of Chinese cabbage and barley showed that inhibitors had strong inhibitory effect on bacteria growth. Overall, the results suggested that VTE and DMY may be potential food preservatives for inhibiting pathogen.