Staphylococcus aureus is an important intramammary pathogen for dairy cows that also is remarkably important for public health. Multiple virulence factors can be involved simultaneously during the pathogenesis of a staphylococcal disease, including adhesion proteins, extracellular enzymes, and toxins. The main objective of this study was to assess virulence factors that are associated with cow intramammary infection (IMI) and of human health concern among Staph. aureus isolates obtained from bulk tank milk (BTM) and adherences on milking equipment surfaces. A total of 166 Staph. aureus isolates from 23 dairy farms were characterized according to their virulence profiles. For virulence factors of importance in IMI, the presence of the virulence markers thermonuclease (nuc) and coagulase (coa) and virulence genes such as fibronectin (fnbA) and intercellular adhesion (icaA, icaD) were assessed. For virulence factors of public health concern, presence of antimicrobial resistance (mecA and mecC) and enterotoxin (sea and seb) genes were analyzed. Among all Staph. aureus isolates, 5 virulence profiles were found; the profile nuc(+)coa(+)fnbA(+)icaA(+)icaD(+)mecA(-)mecC(-)sea(-)seb(-) was the most frequently observed (21 out of 23 dairy farms). No differences were found between the virulence profile frequencies of Staph. aureus from BTM and adherences on milking equipment surfaces. The virulence profiles most frequently observed included genes involved in the adherence and biofilm-forming ability of Staph. aureus, which could represent a potential advantage for the bacterium during the early stages of IMI colonization and for persistence on surfaces. Our results indicate a greater frequency of virulence factors of importance for IMI pathogenesis than virulence factors of public health concern, consistent with the dairy origin of isolates. The mecA, mecC, and seb genes were not observed among Staph. aureus isolates analyzed in this study. However, the sea gene was detected in 3 Staph. aureus isolated from BTM, thus posing a potential public health threat. Our results emphasize the importance of understanding the epidemiology and dynamics of Staph. aureus on dairy farms as a tool for the improvement of udder health and milk safety.