OBJECTIVE Staphylococcus aureus is an important pathogen causing contagious mastitis in cows that need novel treatment rather than antibiotic therapy. This study aimed to investigate the antimicrobial activity of Helicteres isora extracts against S. aureus isolated from subclinical and clinical mastitis cows using an in vitro model. METHODS H. isora pods were extracted using the following solvents: Distilled water, ethanol, acetone, and methanol. The antimicrobial activity of each extract was determined by the disk diffusion method and broth microdilution assay to assess the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). RESULTS The ethanolic extract of H. isora exhibited the largest inhibition zones against S. aureus (31.05±1.20 mm), followed by the aqueous, methanolic, and acetone extracts, respectively (26.34±1.15, 24.23±0.50, and 22.46±1.53). The ethanolic extract also had a strong inhibitory effect on S. aureus, with MIC and MBC of 0.13 and 0.52 mg/mL, respectively. CONCLUSIONS This study revealed that H. isora is a potential alternative natural antibacterial agent against S. aureus infection. The antimicrobial activity of H. isora is most likely mediated by phytochemical constituents.