To mitigate the growth of antibiotic resistance in Staphylococcus aureus due to the misuse of antibiotics， this study aimed to identify natural and safe antimicrobial substances as alternatives to traditional antibiotic treatments for Staphylococcus aureus infections. Six Bacillus strains (Bacillus velezensis SN-20， Bacillus amyloliquefaciens SN-31， Bacillus amyloliquefaciens SN-34， Bacillus amyloliquefaciens SN-36， Bacillus subtilis SN-43， and Bacillus amyloliquefaciens SN-46) were selected for thin-layer chromatography analysis， lipopeptide stability testing， and determination of the antimicrobial mechanism of lipopeptides. The results indicated that these six Bacillus strains could produce lipopeptides such as surfactin， fengycin， and iturin. Among them， the crude lipopeptide extracts from Bacillus velezensis SN-20 and Bacillus amyloliquefaciens SN-46 exhibited significant inhibitory effects on Staphylococcus aureus， with inhibition zone diameters exceeding 12.5 mm and minimum inhibitory concentrations (MIC) of 1.25 mg/mL and 2.5 mg/mL， respectively. The lipopeptides produced by these two Bacillus strains maintained their antimicrobial activity at pH value of 3 and 11， at a temperature of 100 ℃， and after treatment with protease K， demonstrating their excellent and stable antimicrobial properties. Further analysis revealed that the lipopeptides inhibit Staphylococcus aureus through multiple mechanisms， including disrupting cell membrane structure， increasing cell membrane permeability， and inhibiting protein synthesis. These multi-mechanism actions make it difficult for resistance to develop. This study suggested that the lipopeptides produced by Bacillus strains have promising applications and could potentially be developed as new drugs to combat Staphylococcus aureus.