Objective: In this study, the effects of silymarin on the biofilm S. aureus ATCC25923 and its virulence factors were investigated. Methods: The inhibition and clearance of silymarin on S. aureus ATCC25923 biofilm formation were detected by crystal violet staining, the effects of silymarin on its biofilm metabolism were investigated by MTT staining, and the effects of silymarin on its coagulase expression and hemolytic activity were investigated by freeze-dried plasma and aseptic defibrinated sheep blood. In parallel, the effects of different concentrations of silymarin on its biofilm and Fourier transform were observed under an optical microscope. Results: The results showed that the minimum inhibitory concentration (MIC) of silymarin against S. aureus ATCC25923 was 0.5 mg/mL, the inhibition rate of silymarin to S. aureus ATCC25923 biofilm at MIC concentration reached 90%, and the clearance rate reached 89.2%, and the low dose of silymarin could significantly inhibit its hemolytic activity and coagulase expression. Conclusion: The outcomes of the experiment demonstrate the great potential applications of silymarin in the areas of natural preservatives and food safety.