Escherichia coli (E.coli) is highly infectious and harmful, causing serious damage to human health and economic losses. Plant-derived component plumbagin (PLB) is regarded as an alternative to traditional fungicides for its natural, safe, and strong antimicrobial activity. In this study, the antibacterial activity and mechanism of PLB against E.coli were investigated by membrane damage measurement, metabolomics, and molecular docking. Results showed that PLB significantly inhibited the growth and reproduction of E. coli on NA plates and in milk with the minimum inhibitory concentration of 31.25 mg L⁻¹. PLB impaired E. coli cell membrane structure and function, as indicated by disruption of bacterial morphology observed by SEM, increased red fluorescence of PI staining, and higher extracellular conductivity, k⁺, and nucleic acid content. Moreover, PLB treatment increased ROS levels and MDA content, and decreased antioxidant enzyme activities (CAT and SOD), intracellular total lipids, total sugars and total proteins in E. coli. Metabolomics analysis indicated that PLB treatment caused 58 increased and 31 decreased metabolites in E. coli, severely affecting pathways of protein synthesis, lipid metabolism, TCA cycle, ABC transport, etc. Molecular docking showed that PLB has binding potential to key proteases involved in differential metabolite formation in the TCA cycle, glutathione metabolism, and lysine degradation, suggesting that the exertion of antibacterial activity of PLB may be associated with the disruption of these proteins. The above result indicates that PLB inhibited E. coli through membrane damage, and material and energy metabolism disruption, which provides a theoretical basis for PLB as novel preservation.