Antibacterial activity of ethanol extract from Allium prattii against Staphylococcus aureus was analyzed by broth dilution and Oxford cup methods. Effects of ethanol extract from A. prattii on contents of extracellular metal ions and biological macromolecules（i.e. proteins， nucleic acids and polysaccharides）， conductivity of culture medium， integrity of cell walls and ultrastructure of S. aureus were investigated using atomic absorption spectrometry， scanning electron microscopy and so on. Phytochemicals of ethanol extract from A. prattii were quantified by high-performance liquid chromatography. And antibacterial mechanism of ethanol extract from A. prattii was discussed. The minimum inhibitory mass concentration of ethanol extract from A. prattii was 15 mg/mL. After S. aureus was treated with ethanol extract from A. prattii， contents of extracellular K+， Ca2+， Na+， Mg2+， proteins， nucleic acids and polysaccharides markedly increased， and conductivity of culture medium significantly improved. Ethanol extract from A. prattii seriously destroyed cell morphology. Two sulfur compounds （allicin and alliin）， four nitrogen compounds （uridine， β-thymidine， adenosine and 2'-deoxyadenosine monohydrate） and four flavonoids （rutin， kaempferol， quercetin and isorhamnetin） were detected in ethanol extract from A. prattii. Antibacterial activity of ethanol extract from A. prattii against S. aureus was possibly attributed to its phytochemicals such as rutin， quercetin， allicin and kaempferol.