This study sought to analyze the main antibacterial active components of Lactobacillus pentosus (L. pentosus) L-36 cell-free culture supernatants (CFCS) in inhibiting the growth of Staphylococcus aureus (S. aureus), to explore its physicochemical properties and anti-bacterial mechanism. Firstly, the main antibacterial active substance in L-36 CFCS was peptides, which inferred by adjusting pH and enzyme treatment methods. Secondly, the physicochemical properties of the antibacterial active substances in L-36 CFCS were studied from heat, pH, and metal ions, respectively. It demonstrated good antibacterial activity when heated at 65 °C, 85 °C and 100 °C for 10 and 30 min, indicating that it had strong thermal stability. L-36 CFCS had antibacterial activity when the pH value was 2–6, and the antibacterial active substances became stable with the decrease in pH value. After 10 kinds of metal ions were treated, the antibacterial activity did not change significantly, indicating that it was insensitive to metal ions. Finally, scanning electron microscopy, transmission electron microscopy and fluorescence probe were used to reveal the antibacterial mechanism of S. aureus from the aspects of cell morphology and subcellular structure. The results demonstrated that L-36 CFCS could form 1.4–2.3 nm pores in the cell membrane of S. aureus, which increased the permeability of the bacterial cell membrane, resulting in the depolarization of cell membrane potential and leakage of nucleic acid protein and other cell contents. Meanwhile, a large number of ROS are produced and accumulated in the cells, causing damage to DNA, and with the increase in L-36 CFCS concentration, the effect is enhanced, and finally leads to the death of S. aureus. Our study suggests that the main antibacterial active substances of L-36 CFCS are peptides. L-36 CFCS are thermostable, active under acidic conditions, insensitive to metal ions, and exhibit antibacterial effects by damaging cell membranes, DNA and increasing ROS. Using lactic acid bacteria to inhibit S. aureus provides a theoretical basis for the discovery of new antibacterial substances, and will have great significance in the development of antibiotic substitutes, reducing bacterial resistance and ensuring animal food safety.