The study was intended for identification and characterization of Staphylococcus aureus isolated from raw cow milk. A total of 47 milk samples were collected from Sheshmore, Shutiakhali and Bangladesh Agricultural University Dairy Farm, Mymensingh. Using bacteriological, biochemical and PCR-based identification schemes, 12 (25.53%) isolates were confirmed as S. aureus. All the isolates showed β-hemolysis on 5% sheep blood agar. S. aureus specific nuc gene (target size 279-bp) was amplified in the cases of all isolates. The isolates were found as resistant to Penicillin (100%), Erythromycin (75%) and Amoxicillin (100%). On the other hand, the isolates were sensitive to Ciprofloxacin (83.33%), Oxacillin (100%), Cloxacillin (100%) and Neomycin (100%). The isolated S. aureus showed increased... resistance to broad spectrum antibiotic (e.g., Ciprofloxacin). As many people have a tendency to drink raw milk and raw milk products, there is high risk of S. aureus infection in human.