Bovine mastitis remains the largest hazard in the global dairy industry and has facilitated the development of various therapeutic strategies. Silver is a well-known disinfectant that is widely used in the treatment of clinical disease. In this study, we separated bovine mammary gland epithelial cells (BMEC) using an enzyme probe. We also examined safe concentrations for the application of silver ions in bovine mastitis, particularly in cases induced by Staphylococcus aureus. S. aureus-derived alpha-toxins induced cell damage through DNA fragmentation, reactive oxygen species (ROS) generation, and the dissipation of mitochondrial transmembrane potential (MTP) in BMEC. Silver ion treatment doses of lower than 2 ppm did not induce BMEC damage, but silver ion concentrations greater than 4 ppm was accompanied by DNA fragmentation. Furthermore, silver ions doses below 2 ppm inhibited alpha-toxin-induced cell damage through the reduction of ROS generation. Recognizing this, it demonstrate that low doses of silver ions inhibit alpha-toxin-induced BMEC damage and suggest that silver ions may be a potentially beneficial treatment against bovine mastitis, particularly in cases induced by S. aureus.