To elucidate the key antibiofilm components of Zanthoxylum armatum DC. essential oil (ZAEO) against Bacillus amyloliquefaciens and their associated intracellular mode of action, the PharmMapper server was used to screen potential targets and these predicated targets were then refined by genome annotation information, to predict and select the key active components of ZAEO and action targets involved in antibiofilm. The interaction network between ZAEO's active components and potential targets was constructed by using Cytoscape software. The GO function and KEGG pathway enrichment analysis were further performed using the String database. The core targets were obtained through potential target interactions analysis by STITCH database and performed network topology was analysed by Cytoscape software. Additionally, the CB-DOCK2 web server was used to carry out the molecular docking between the active components and the core targets. Finally, the in vitro antibiofilm activity of key components of ZAEO was assessed through culture experiments involving crystal violet staining and bacterial motility test. The results showed that a total of 21 active ZAEO components, 133 potential targets and 8 core targets were obtained by network pharmacology analysis. GO enrichment analysis yielded a total of 17 subcategories, including 6 subcategories related to biological processes, 8 subcategories related to molecular functions, and 3 subcateories related to cellular components. KEGG pathway enrichment analysis revealed 8 signaling pathways, including secondary metabolite biosynthesis, amino acid biosynthesis and other pathways. Molecular docking analysis demonstrated that three key active components, namely uncineol, farnesol acetate and trans-farnesol, exhibited strong binding affinity to the core target OdhA, CarB and PurF through intermolecular forces such as hydrogen bonds and hydrophobic interactions, forming stable complex conformation. Furthermore, the in vitro biofilm culture verification experiment confirmed that the range of 0.32 to 1.28μL/mL of farnesol acetate and trans-farnesol displayed significant antibiofilm effects against Bacillus amyloliquefaciens. These results aimed to provide new technical routes for the intracellular molecular mechanism of the antibiofilm of ZAEO.