Quaternary ammonium compounds (QAC) are widely used biocides in the field of medical and veterinary practice. Resistance to QACs is an emerging problem due to this widespread use. The present study was undertaken to detect the presence of genes that contribute to resistance to QACs in Staphylococcus aureus isolates obtained from udder surface and mastitis milk. A total 22 isolates of S. aureus were obtained from udder washings and milk of bovine mastitis cases. Among these, seventeen isolates were from udder wash samples and five isolates from clinical and subclinical cases of mastitis. Broth microdilution assay was performed to assess the minimum inhibitory concentration (MIC) of the isolates against Cetyltrimethylammonium bromide (CTAB), a QAC. Polymerase chain reaction (PCR) was done targeting qac A/B gene which codes for efflux pump which targets QACs. In the present study qac A/B gene was detected in seven out of 22 isolates of S. aureus. Three of these isolates were obtained from udder washings before milking and four, after milking. None of the S. aureus isolates from mastitis milk possessed the gene. The average minimum inhibitory concentration of qac A/B positive isolates against CTAB was 0.63 ± 0.55 μg/ml. The average MIC between qac A/B positive and negative isolates were statistically insignificant. The study points to the fact that multiple factors could be contributing to biocide resistance in S. aureus.