Abstract: The aim of the study was to evaluate the combined effects of rosmarinic acid and ε-polylysine, and the investigate synergy of this compounds against Staphylococcus aureus. The antibacterial effects were analyzed by the minimal inhibitory concentration (MIC), inhibition zone, synergistic action, antibacterial activities, soluble protein, reducing sugar leakage and bacterial surface protein SDS-PAGE (sodium dodecylsulphate polyacrylamide gel electrophoresis). Results showed that the MIC of rosmarinic acid and ε-polylysine to Staphylococcus aureus was 4 and 0.1875 mg/mL, respectively, and the FICI (fractional inhibitory concentration index) was 0.5, which had a good synergistic effect. 1/4 MIC rosmarinic acid and 1/4 MIC ε-polylysine had strong antibacterial activity. They could damage the cell membrane of bacteria, lead to the leakage of protein and reduce sugar, and the molecular weight of the protein on the surface of bacteria changed by protein profile analysis showing that these two antibacterial ingredients promoted the degradation of the bacteria surface protein, then inhibited the toxicity of virulence factors on the surface of Staphylococcus aureus.