Mastitis remains the most frequent and the most expensive disease of dairy breeding. The objective of the study was to study S. aureus isolates collected from sub-clinical bovine mastitis in the Tiaret region, Algeria, by determining their antimicrobial susceptibility and their virulence traits. Sixty-two S. aureus isolates collected from subclinical bovine mastitis were studied by determining their antimicrobial susceptibility according to CLSI guidelines and nine genes encoding virulence factors and resistance to methicillin and penicillin were determined by PCR. Multi-drug resistance was observed in 19 (30.64%) isolates and five (8%) were methicillin-resistant S. aureus (MRSA), four of them harbored the mecA gene; however, mecC gene was not detected. Out of 59 penicillin resistant isolates, 14 harbored the blaZ gene; one of them co-harbored the mecA gene. The following virulence genes were detected: eta (n=23; 37%), icaA (20; 32.2%), icaD (18; 29%), etb (16; 25.8%), luk E-D (14; 22.5%), and sea (6; 9.6%). The tsst-1, lukF/lukS, luk-M genes were not detected. The occurrence of MRSA and multi-drug resistant (MDR) S. aureus isolates as well as genes encoding virulence factors playing an important role in the pathogenesis of sub-clinical bovine mastitis and of harmful potential to human is a cause for concern.