Abstract: Staphylococcus aureus is one of the most common pathogens of infectious diseases with a significant morbidity and mortality.Antimicrobial activity of azithromycin against S.aureus were determined for each microorganism grown in suspension or biofilm using microbroth dilution method,and influence of azithromycin on biofilm formation and secretion of virulence factors by S.aureus was assessed by biofilm formation assay,hemolysin assay,Western blot and real-time RT-PCR.The results showed that azithromycin had better antimicrobial activity against S.aureus grown in suspension or biofilm,and effective inhibition of biofilm formation;subinhibitory concentration of azithromycin reduced the production of α-haemolysin and enterotoxin A by S.aureus in dosedependent manner.In particular,1/2MIC of azithromycin almost completely suppresses the production of α-haemolysin and enterotoxin A.This study might be especially helpful for the treatment of S.aureus infections.