Summary The purpose of the study was to explore the antibacterial effect and mechanism of a novel bacteriocin of Lactiplantibacillus plantarum (L. plantarum) against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The antibacterial activity was determined by the Oxford cup method, and the dynamic growth curves were conducted with continuous shaking incubation. The result showed that the bacteriocin was a protein or protein‐like antibacterial substance susceptible to pepsin and trypsin. And it had good antibacterial activity, pH stability, thermostability and enzyme treatment stability against E. coli and S. aureus. The SEM, flow cytometry and nucleic acid leakage showed that the bacteriocin disrupted the cell structures of the two bacteria by damaging cell walls and cell membranes. An agarose gel electrophoresis and SDS‐PAGE analysis showed that the bacteriocin inhibited DNA replication and interfered with the protein formation, which resulted in the inhibition of the two bacteria's growth. Therefore, the use of the L. plantarum bacteriocin might be a promising biocontrol strategy to inhibit the pollution of E. coli and S. aureus simultaneously in foods.