Staphylococcus aureus is important pathogenic bacteria in both humans and animals. In animals, S. aureus is the main causative agent of mastitis and 30% of bovine mastitis cases are associated with S. aureus. Bovine mastitis is the most devasting disease in the dairy industry. The treatment, which includes the administration of antibiotics, has been only partially successful, this is due to the development of antibiotic resistance, and the ability of the bacteria to invade and hide in the host cells. Therefore, the present study aims to investigate the antibacterial activities of the Ikarugamycin compound against intracellular S. aureus, and to determine the cytotoxicity of the compound toward bovine mammary epithelial cells ( Mac-T cells ). Minimum inhibitory concentration (MIC) was used to determine the antibacterial activity of Ikarugamycin. In-vitro infection models’ assay was used to infected Mac-T cells with S. aureus. Ikarugamycin intracellular antibacterial activity assays were used to determine the bactericidal activity of Ikarugamycin against intracellular S. aureus. Alamar blue assay was used to evaluate the cytotoxicity of Ikarugamycin against Mac-T cells. S. aureus is susceptible to Ikarugamyicin with MIC value 0.6 μg/mL. Ikarugamycin at 4 MIC and 8MIC have bactericidal activity by reducing 3 and 5 log 10 CFU/ml of S.aureus in the first six-hour respectively. Moreover, the Ikarugamycin possesses intracellular antibacterial activity, at 5 μg/mL killed 90% of intracellular S. aureus. The concentration of Ikarugamycin that inhibits 50% of Mac-T cells (IC50) was 9.2 μg/mL, therefore the IC50 is far from the concentration required to kill 90% of intracellular S. aureus in Mac-T cells. The study highlighted that Ikarugamycin antibiotic can be used to deal with infections cause by intracellular and multi-drug resistance S. aureus in case of mastitis