Staphylococcus aureus is one of the most prevalent pathogens, and a causative agent of a variety of infections in humans and animals. A total of 48 semen samples were collected from healthy bucks of different breeds to investigate the prevalence of S. aureus . Antimicrobial resistance and virulence of the Staphylococcus isolates were determined to assess the adverse effects of them on buck fertility. The bacterial isolates were tentatively confirmed as Staphylococcus spp. based on the Gram’s staining, growth on Mannitol salt agar and catalase test. Overall, 75% (n = 36) of the samples were positive for Staphylococcus spp. from the total 48 buck semen ejaculates from different breeds. Out of 36 staphylococcal isolates, 23 (47.92%) were coagulase negative (CoNS) and 13 (27.08%) were coagulase positive Staphylococcus (CoPS) based on the slide coagulase test. In the current study, on the basis of molecular characterization, we identified S. aureus , S. chromogenes, S. haemolyticus, S. sciuri, S. simulans and S. epidermidis amongst the staphylococcal isolates in the buck semen. This study revealed a high prevalence of Staphylococcus species in semen of the healthy bucks. The isolates exhibited varying degrees of multidrug resistance genotypically as well as phenotypically. The presence of antibiotic resistance and virulence genes may pose a potential threat to reproductive health of animals, highlighting the need for vigilant monitoring of these isolates at the time of semen cryopreservation.