This study explored the antibiofi lm effi cacy of 3,3’-diindolylmethane (DIM) on Staphylococcus aureus and its disinfection on common food-contact surfaces. The minimum biofilm inhibitory concentration (MBIC) of DIM on S. aureus was 62.5 μmol/L, while it did not impede the bacterial growth evaluated by growth curve and XTT reduction assay. DIM in the concentration range of 31.2?62.5 μmol/L demonstrated a dose-dependent antibiofilm activity to S. aureus, as confirmed by light microscopic (LM), confocal laser scanning microscopic (CLSM), and scanning electron microscopic (SEM) analyses. At DIM of 62.5 μmol/L, the biomass of S. aureus biofi lm was signifi cantly reduced by 97% and its average thickness by 58% (P < 0.05). DIM of 62.5 μmol/L inhibited the bacterial initial adhesion and proliferation, as well as cell motility; the release of extracellular DNA (eDNA) and extracellular polysaccharide (EPS) were reduced by 75% and 69%, respectively. DIM exhibited a strong inhibition to S. aureus biofi lm formation on common food-contact surfaces, including 304 stainless steel, glass, and polyvinyl chloride (PVC) but not disperse the mature biofi lm. Overall, our investigation identifi ed DIM as a promising antibiofi lm agent and its suitability to prevent the biofi lm formation of S. aureus on common food-contact surfaces utilized during food processing.