Abstract:[Background] As a common food-borne pathogen, Staphylococcus aureus can do harm to food safety and human health. Although chemical preservatives can inhibit the growth of food-borne pathogens, they pose a threat to human health and the environment. [Objective] To decipher the mechanism of silymarin in inhibiting S. aureus ATCC 25923 for the development of plant-derived bacteriostatic agents. [Methods] We investigated the effects of silymarin on the membrane structure of bacteria by measuring alkaline phosphatase, β-galactosidase activities, and PI staining. The effects of silymarin on the macromolecules of bacteria were explored by DNA electrophoresis and determination of DNA and protein concentrations in bacteria cells. Furthermore, the effects of silymarin on the synthesis of polysaccharide intercellular adhesion (PIA), bacteria in biofilms, and the proteins and polysaccharides in mature biofilms were explored. Fourier transform infrared spectrometry was employed to reveal the changes of bacterial composition. On the basis of the results, the diversity of the mechanisms of silymarin in inhibiting S. aureus ATCC 25923 was expounded. [Results] The minimum inhibitory concentration (MIC) of silymarin against S. aureus ATCC 25923 was 0.5 mg/mL. Silymarin can inhibit the growth of bacteria by destroying the membrane structure of bacteria cells, reducing the concentrations of DNA and protein, Inhibit the synthesis of PIA in biofilm, and degrading the mature biofilm components. [Conclusion] Silymarin can inhibit the growth of S. aureus in multiple ways, which is expected to provide a theoretical basis for the further application of natural products from plants.