Different molecular weights of ε-PL have different antibacterial activities， but the mechanism that causes this difference is not clear. In this study， Gram-positive bacteria Staphylococcus aureus was selected as the research object. From the aspects of antibacterial activity， metabolic level， and cell wall， scanning electron microscope （SEM）， microplate reader， gas chromatography-mass spectrometry （GC-MS） and other instruments analyzed the difference in bacteriostatic activity caused by different molecular weight ε-PL， and explored the mechanism of different molecular weight ε-PL on S. aureus. The experiment results showed that compared with the low-molecular-weight（<1 ku） ε-PL treatment group， the high-molecular-weight ε-PL（1-3 ku and >3 ku） makes the surface of the cells sag and shrink to different degrees. The metabolomics method was used to study the differences in the metabolic levels of S. aureus after the action of different molecular weight ε-PL. The results showed that high molecular weight （1-3 ku and > 3 ku） ε-PL and the initial ε-PL inhibited the glycolytic pathway and tricarboxylic acid cycle much more than the low molecular weight （<1 ku） ε-PL. It has been verified that the bacteriostatic effects to S. aureus with different molecular weight ε-PL are different， which provides theoretical support for the use of preservatives in actual food processing and production processes.