BACKGROUND: Subclinical mastitis plays an important role in the economic losses of dairy cattle farms. Staphylococcus aureus is one of the most important causes of this disease. Treatment of this disease with synthetic antibiotics has complications like antibiotic resistance. Using herbal antibiotics can be an excellent way to reduce these side effects.OBJECTIVES: This study aimed to determine the minimum inhibitory concentration of alcoholic extract of olive leaf on Staphylococcus aureus isolated from milk of cows with subclinical mastitis to achieve herbal treatment.METHODS: This study was conducted on 175 Holstein female cattle. The milk samples of 60 cows were obtained with the sterilized method, and Subclinical mastitis-positive cases were determined using the California mastitis test. Staphylococcus aureus bacteria were isolated from positive samples by culture method, and the minimum inhibitory concentration of olive (Olea europaea L.) leaves alcoholic extract on isolated bacteria was determined by microdilution method.RESULTS: From 175 cows under study, 60 cows had a positive California mastitis test, and Staphylococcus aureus separated from milk samples of 14 cows. The minimum inhibitory concentration of olive (Olea europaea L.) leaves extract on this bacterium was 12000 ppm.CONCLUSIONS: Alcoholic extract of olive (Olea europaea L.) leaves has an antimicrobial effect on Staphylococcus aureus as a cause of mastitis. The minimum concentration required for this effect was 12000 ppm. Further studies on the impact of this plant on other bacterial causes of subclinical mammary inflammation in cows and investigation of the effective substances in the extract are needed.