Biofilm-forming Staphylococcus aureus can easily accumulate on various food contact surfaces which induce cross-contamination and are difficult to eliminate in the food industry. This study aimed to evaluate the anti-biofilm effects of natural product biochanin A against S. aureus. Results showed that biochanin A effectively eradicated established S. aureus biofilms on different food-contact materials. Fluorescence microscopic analyses suggested that biochanin A disintegrated the established biofilms by dissociate extracellular polymeric substance (EPS) in matrix. In addition, biochanin A at the sub-MIC concentration also effectively inhibited the biofilm formation by regulating the expression of biofilm-related genes (icaA, srtA, eno) and suppressing the release of EPS in biofilm matrix. Molecular docking also demonstrated that biochanin A conducted strong interactions with biofilm-related proteins (Ica A, Sortase A, and Enolase). These findings demonstrated that biochanin A has the potential to be developed as a potent agent against S. aureus biofilm in food industries. KEY POINTS: • Anti-biofilm effect of biochanin A against S. aureus was revealed for the first time. • Biofilm of S. aureus on various food-contact surfaces were efficiently eradicated. • Biochanin A prevented S. aureus biofilm formation via reducing EPS production.