Background Staphylococcus aureus ( S. aureus ) has been reported as the most commonly isolated highly contagious pathogen from human, animals and animal products. Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a significant pathogen with zoonotic potential that could have devastating consequence for the health and well-being of animals and human. Methods A cross-sectional study was conducted from July 2020 to January 2021. A total of 233 samples from cow milk, udder swabs and milkers’ hand swabs were collected for culture and identification based on the standard protocol. Antimicrobial susceptibility tests were performed for all isolates by using Kirby Bauer’s disk diffusion test. MRSA was detected by cefoxitin disk diffusion test. Results S. aureus was isolated from 50 (21.46%) of 233 samples and the prevalence of MRSA was 4%. The highest prevalence was found in cow milk 36 (25.53%) followed by hand swabs 10 (19.23%) and udder swabs 4 (10%). S. aureus prevalence was 58.33%, 30.0%, 21.43%, 17.92%, 15.79% in farm D, C, E, A, B respectively. A large percentage (58.33% and 30%) were from farm D and C. S. aureus isolation rate showed statistically significant association with farm types (p = 0.011) and with previous mastitis exposure (p = 0.001). High level of resistance was observed to penicillin (94%) and ampicillin (92%), but low level resistance to gentamicin (0%), amikacin (0%), ceftriaxone (0%), chloramphenicol (4%), ciprofloxacin and cefoxitin (4%). The overall prevalence of multidrug resistance (MDR) was 10.42%. Conclusion Prevalence of S. aureus in milk showed statistically significant association with respect to previous mastitis exposure and farm types (p = 0.011). High level of resistant to penicillin and ampicillin was observed. Therefore, effective mastitis control programs, best veterinary practice among all farms and use of antibiotics in the farm should be strictly controlled.