[Background] Staphylococcus aureus is a type of common food-borne pathogenic bacteria that can easily form biofilm on the surface of food and processing equipment, leading to food corruption and disease spread, and threatening food safety. [Objective] To study the inhibition of S. aureas biofilm formation by oridonin. [Methods] The inhibition of biofilm formation by oridonin was studied using crystal violet staining assay and scanning electron microscopy. The influence of oridonin on the formation of polysaccharide intercellular adhesion and the release of extracellular DNA (eDNA) was detected by Congo red agar and spectrophotometer. RT-PCR analysis was used to determine the effect of oridonin on the expression of genes, including icaA, cidA, agrA and sarA. [Results] Oridonin showed strong antimicrobial activity on S. aureas biofilm formation. Polysaccharide intercellular adhesion formation and eDNA release were greatly inhibited by oridonin. eDNA release decreased by 48.62% after incubated with oridonin at 1/4MIC for 16 h. Oridonin could significantly inhibit the expression of biofilm forming related genes in S. aureas. After incubated with of oridonin at 1/2MIC for 16 h, the relative expression of icaA, cidA, agrA and sarA of S. aureas were reduced by 91.6%, 94.7%, 77.6% and 70.4%, respectively. [Conclusion] Oridonin can significantly inhibit the biofilm formation of S. aureas via the reduction of icaA and cidA expression, so as to influence the synthesis of polysaccharide intercellular adhesion and the eDNA release.