[Background] Lactic acid bacteria, which are widely used in many industries such as food and feed, have become research hotspots for making biological preservatives. [Objective] The antibacterial substance of Lactobacillus plantarum DY6, with the excellent antibacterial effect, was preliminarily explored, which would provide a reference for further application. [Methods] The physicochemical properties of the antibacterial substances in the fermentation broth of Lactobacillus plantarum were studied. The GC-MS metabolomics method was used to analyze the metabolites in the fermentation supernatant, while the main inhibitory substances were speculated by multivariate statistical analysis and initially separated by semi-preparation HPLC for further identification by GC-MS. [Results] Lactobacillus plantarum DY6 had strong inhibitory effect on Staphylococcus aureus, Escherichia coli and Salmonella. The antibacterial ability of fermentation supernatant with different fermentation time was tested. The supernatant of 0?4 h had no antibacterial activity, and the antibacterial ability was gradually increased after 8 h. The antibacterial activity of fermentation supernatant tended to be stable from 24 h to 48 h and was the best at 48 h. The antibacterial diameter was 15.28 mm. The differential markers of lactic acid bacteria fermentation broth were analyzed by multivariate statistical analysis. The main differences were found to be organic acids (such as lactic acid, acetic acid, propionic acid, etc.) and fatty acids (such as caprylic acid and citric acid). Antibacterial components of fermentation supernatant were obtained via semi-preparation HPLC, mainly including: organic acids (such as lactic acid, acetic acid, 3-phenyl lactic acid, phenylpropionic acid, etc.), fatty acids (such as citric acid, octanoic acid, citric acid, etc.), in addition to a small amount of aldehydes and alcohols. [Conclusion] It was determined that the antibacterial substances of Lactobacillus plantarum DY6 were mainly organic acids and fatty acids, which provided a theoretical basis for further antiseptic applications.