Biocides are commonly used in infection control programs to prevent the new infections of the udder and colonization of microorganisms on other contact surfaces. In the present study, a multiplex PCR (mPCR) assay simultaneously amplifying 04 biocide resistance genes was standardised. The mPCR was applied on 30 S. aureus isolates, isolated from milk of mastitis affected cows and prevalence of these genes in S. aureus was studied. High resistance mediated by sepA (83.33%), norA (80%) and mepA (80%) resistance genes was observed while resistance mediated by qacA/B (3.33%) gene was found very low. Genetic resistance against biocides is a widespread problem that has been extensively studied in food industry and hospitals particularly targeting methicillin resistant S. aureus, however, limited studies on biocide resistance in S. aureus of animal origin have been undertaken.