Abstract: To probe into the antibacterial effects of Lactiplantibacillus plantarum 414 fermentation supernatant against common foodborne pathogens and its material basis, this study employed the Oxford cup method, micro-broth dilution technique, scanning electron microscopy (SEM), and metabolomics analysis. As evidently demonstrated by our research findings, the fermentation supernatant considerably inhibited Staphylococcus aureus, Salmonella, Escherichia coli O157:H7, and Shigella, with inhibition zone diameters of 20.09±1.15、15.43±0.71、15.84±1.53, and 19.15±1.85 mm, respectively, with a minimum inhibitory concentration (MIC) of 2.5 mg/mL for all strains. As SEM observation suggested, the supernatant disrupted the integrity of indicator bacteria cell membranes. pH neutralization experiments confirmed organic acids as the primary antibacterial components. Metabolomics and targeted quantitative analysis further identified the major organic acids with antibacterial potential in the supernatant as lactic acid, citric acid, oxalic acid, malic acid, succinic acid, α-ketoglutaric acid (AKG), fumaric acid, and pyruvic acid, with concentrations of 9.582±0.093, 1.393±0.037, 0.391±0.009, 0.077±0.0046, 0.072±0.0026, 0.026±0.0017, 0.007±0.0005, and 0.003±0.0002 mg/mL, respectively. To sum up, the fermentation supernatant of L. plantarum 414 exerts antibacterial effects by disrupting the cell membranes of indicator bacteria, with its activity likely attributable to a complex system encompassing multiple organic acids, represented by high-concentration lactic acid. This study provides a preliminary analysis of the antibacterial substance basis of this strain at the organic acid metabolism level, thereby offering theoretical support for its development as a biological preservative.