The objectives of the study were to detect toxins and antiseptic resistance genes in Staphylococcus aureus isolated from cows with subclinical mastitis in Egypt. A total of 400 quarter milk samples (QMS) were collected from different dairy herds in which quaternary ammonium compounds (QAC) had been used as a disinfectant for more than 3years. The collected samples were subjected to bacterial investigation. S. aureus was successfully isolated confirmed by duplex PCR targeting 16S rRNA and nuc genes. It also determined their antibiogram and sensitivity to disinfectant. Genes of QAC(qacA/B), enterotoxins (Sea, Seb) and exfoliative toxins (ETB) were detected by simplex and multiplex PCR. Results of bacterial investigation revealed 103 (25.75%) S. aureus isolates. Results of the antibiogram demonstrate that most microbial antibiotic resistance were recorded for Penicillin G (85.7%) and Tetracycline (54.2%). While Gentamycin, Neomycin and Amoxicillin+ clavulanic acid show moderate resistance (21.4%, 10%, and 7.1%) respectively, although Norfloxacin and Cephradine exhibited seldom resistance with high sensitivity of 95% and 94.3% respectively. Regarding the results of QAC sensitivity, only 8 isolates (7.76%) were resistant to benzalkonium chloride (BC) versus to 13 isolates (12.62%) harbour QAC gene could be detected by PCR with specific amplicon of 220bp corresponding to qacA/B. The results revealed Positive amplification of 102 bp specific for Sea gene in 19(18.44%) isolates and 164bp specific for Seb gene in 13(12.62%) isolates while there is no amplification was detected for etb gene. In conclusion, an Antibiogram, as well as the identification of toxigenic and QAC genes in this study, may open another perspective in planning some alternative therapeutic strategies against multi resistances S. aureus mastitis. Monitoring cross-resistance between antibiotics and antiseptic should be further investigated.