Lameness & mastitis are recognized as one of the most important disease affecting the broiler chicken and dairy cattle respectively. The study was performed to observe the biochemical characters and antibiotic susceptibility of Staphylococcus aureus (S. aureus) causing lameness in broiler chickens and mastitis in cows. Characters of the isolates were determined by biochemical tests such as mannitol fermentation, catalase, coagulase test. To investigate the antibiogram profiling, frequently used antibiotic discs were tested. From this study, it was observed that at least 84% isolates of S. aureus from lame broiler chicken and mastitic cow’s milk sample showed positive result in mannitol fermentation test, catalase and coagulase test respectively. But all isolates showed negative result in oxidase and motility test. Interestingly all the bacterial isolates from both broiler and milk sample showed different antibiogram profiling. Antibiotic susceptibility testing suggested that approximately more than 45-65% of the isolates were resistant to penicillin and about 25% resistant to amoxicillin in all isolates. The study reveals that more than 32% isolates were susceptible to methicillin resistant S. aureus (MRSA) and 35% chicken isolates were vancomycin resistant. Maximum resistance was observed against azithromycin (68%) of mastitic cow isolates. The present study also showed that S. aureus was most sensitive to ciprofloxacin (86-100%). Based on results of this study we conclude that drug resistance of S. aureus may vary with the antibiotics being used and with different population.