Staphylococcus aureus is one of the most common causes of mastitis worldwide. This study aimed to determine the prevalence and antimicrobial resistance (AMR) patterns of S. aureus in mastitic milk samples collected from camel farms in Matrouh Governorate, Egypt. A total of 200 mastitic camel milk samples were evaluated for S. aureus using both conventional culture-based and molecular-based methods. Antibiotic susceptibility testing of S. aureus isolates was conducted using disc diffusion and agar dilution methods, with antibiotic resistance genes identified through polymerase chain reaction with specific primers. Out of samples tested, 60 (30.00%) were positive for S. aureus. The isolates displayed the highest of resistance against piperacillin-tazobactam (55.00%) followed by trimethoprim-sulfamethoxazole (45.00%) and amoxicillin (40.00%). Half of the isolates were multidrug-resistant (MDR). The AMR genes included methicillin-resistant gene (mecA), ß-lactamase gene (blaZ), tetracycline resistance gene (tetK), erythromycin resistance gene (ermB) and vancomycin resistant gene (vanA) were detected in 100%, 100%, 95.00%, 90.00% and 20.00% of the isolates, respectively. In conclusion, the presence of MDRS aureus as a cause of clinical camel mastitis is a significant veterinary and public health concern. These findings highlight the importance of proper antibiotic use in Egyptian camel farms and the need for molecular techniques to fully understand the genetic profile of antimicrobial-resistant S. aureus isolates. [ABSTRACT FROM AUTHOR]