This study aimed to investigate the chemical constituents and antibacterial mechanism of Bletilla striata (Thunb.) Reichb.f.against Staphylococcus aureus.A comprehensive chromatographic separation was conducted to isolate and purify active compounds from B. striata,then all the isolated compounds were identified by NMR spectra.The inhibitory effect of the isolated compounds on S. aureus was evaluated via broth microdilution.The antibacterial mechanism of the active ingredients in B. striata was explored from several aspects including the growth curves of S. aureus,extracellular alkaline phosphatase content,DNA leakage,cell wall and cell membrane integrity.Ten compounds (1-10) were isolated and identified as shancidin (1),coelonin (2),lusianthridin (3),orchinol (4),isoshancidin (5),blestriarene A (6),(E)-3-(4-hydroxyphenyl)acrylic acid (7),3-phenylpropanoic acid (8),ethyl 2-(4-hydroxyphenyl)acetate (9),ethyl 2-(4-ethylphenyl)acetate (10).Besides, compounds 7-10 were isolated from B. striata for the first time. Furthermore, compound 6 exerted an excellent bacterial inhibitory effect with a minimum inhibitory concentration of 10 μg/mL (positive control amoxicillin 0.8 μg/mL).The antibacterial mechanism results indicated that compound 6 disrupted the bacterial membrane structure,leading to alkaline phosphatase and DNA leakage,which resulted in bacterial death. Compound 6 may exert antibacterial activity by destroying cell wall and cell membrane.