Staphylococcus aureus is a common foodborne pathogen, and its prevention and control has always been a hot topic of concern. The purpose of this study was to screen bacteriocin-producing lactic acid bacteria (LAB) and explore its antibacterial and antibiofilm activity against S. aureus. A total of 67 strains of lactic acid bacteria were isolated from fermented foods, and 9 strains with good antibacterial activity against S. aureus and Escherichia coli were screened out according to the inhibition zone experiment and identified by 16S rDNA sequencing. After acid neutralization, and catalase and protease treatment, it was found that Lactiplantibacillus plantarum 65 could produce a new bacteriocin. The cell-free supernatant (CFS) of L. plantarum 65 was separated and purified by organic solvent extraction, sephadex LH20, and reversed-phase high-performance liquid chromatography (RP-HPLC). The antibacterial active peptide obtained was a new type of bacteriocin with an amino acid sequence of KVKPLHA and a molecular weight of 791.5Da. The CFS of L. plantarum 65 showed antibiofilm activity against S. aureus and Listeria monocytogenes, with the highest inhibition rate reaching 83.07%. L. plantarum 65 isolated from fermented food products could produce novel bacteriocin with the ability to inhibit growth and biofilm formation of S. aureus.  IMPACT STATEMENT: The obtained results highlight the potential of bacteriocin-producing L. plantarum 65 have dual potential for antibacterial and antibiofilm activity against S. aureus, and it can be used for the prevention and control contamination of S. aureus in food.