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Ethnopharmacological Relevance: Hypericum ascyron L. (H. ascyron L.) has been used as a traditional medicine for the treatment of wounds, swelling, headache, nausea, stomach ache, abscesses, dysentery and chronic bronchitis. Pharmacological studies are necessary to provide a scientific basis to substantiate their traditional use. In this study, antimicrobial effect and its mechanism of the H. ascyron L. extract was investigated., Materials and Methods: Antimicrobial activity of the H. ascyron L. extract was evaluated by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT). To confirm the antimicrobial mechanism of H. ascyron L. extract on bacterial cells, Annexin V/propidium iodide (PI) double staining, TUNEL, and DNA laddering analysis were performed., Results: Antibacterial effects of the H. ascyron L. extract were tested on Enterobacter cloacae, Klebsiella pneumonia, Escherichia coli, Staphylococcus aureus and Micrococcus luteus, and the results showed that the sensitive bacteria of H. ascyron L. extract mainly included E. coli, S. aureus and M. luteus, especially M. luteus. The MBC value of H. ascyron L. extract on M. luteus was equal to the MIC (20mg/mL). H. ascyron L. extract could induce cells death of M. luteus through apoptosis pathway., Conclusions: This study provides scientific support for some of the traditional uses and the pharmacological activities of H. ascyron L. Our data provide a rational base for the folkloric use of H. ascyron L., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)