Biofilm formation by Staphylococcus aureus on food contact surfaces is one of the most important issues for the food safety. The difficulties in controlling biofilms have driven the search for new antibacterial and antibiofilm agents from natural resources. The aims of the present study were to investigate the antibacterial and antibiofilm activities of the methanolic extract from Zanthoxylum bungeanum Maxim. leaves and identify the active compounds. By bioassay guide of inhibitory activity against S. aureus, four antibacterial compounds were separated from this extract and identified as neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and 4-O-caffeoyl-2,3-dihydroxy-2-C-methylbutyric acid based on MS and NMR data analyses. The four compounds exhibited moderate antibacterial activity against S. aureus with minimum inhibitory concentration of 5 mg/mL. Moreover, a fraction consisted of the four compounds was subjected to antibiofilm assays against S. aureus. Crystal violet staining and XTT reduction assay demonstrated that this fraction showed an excellent inhibitory efficacy on the biomass and metabolic activity of S. aureus biofilm. Scanning electron microscopic observation displayed that this fraction induced severe morphological changes in the architecture of S. aureus biofilm, which further confirmed that it possessed a potent inhibitory activity on the biofilm formation of S. aureus. So, these results suggested that Z. bungeanum leaves could be used as an attractive and promising candidate for the development of natural antibacterial agent for controlling food-related bacterial biofilms.