Staphylococcus aureus is one of the most common pathogens in bacterial biofilm infections. Antibiotic treatment for infection caused by S. aureus biofilms is challenging, and few effective strategies have been developed to combat these infections. The aim of this study was to investigate the effect and possible mechanisms of artesunate on the biofilm formation of S. aureus. Bacterial growth curves were determined by a microtiter plate. Biofilm formation was determined by the crystal violet staining method and confocal laser scanning microscopy. Bacterial adhesion was assayed by the colony-counting method. The expression of virulence and adhesion genes was determined by real-time PCR. The hemolytic activity and expression of ɑ-hemolysin were analyzed using rabbit erythrocytes and Western blotting. The results showed that artesunate could significantly inhibit the biofilm formation of S. aureus in a dose-dependent manner. Artesunate could also inhibit bacterial adhesion and the expression of hla, RNAIII and agrA as well as ɑ-hemolysin production. The effect of artesunate on adhesion genes (clfA, clfB, fnbA, fnbB) had strain specificity, but it did not affect the expression of ica genes. The results indicated that artesunate might inhibit ɑ-hemolysin synthesis by the agr system, which inhibits biofilm formation.