Background: The diffusion of antimicrobial resistance is a significant concern for public health worldwide. Staphylococcus aureus represents a paradigm microorganism for antibiotic resistance in that resistant strains appear within a decade after the introduction of new antibiotics. Methods: Fourteen S. aureus isolates from human specimens and twenty-one from samples of animal origin, were compared for their antimicrobial resistance and biofilm capability. In addition, they were characterized at the molecular level to detect the antimicrobial resistance mecA gene and genes related with enterotoxin, toxin, and biofilm production. Results: Both phenotypic and molecular analysis showed main differences among human- and animal-derived isolates. Among the human-derived isolates, more multidrug-resistant isolates were detected and mecA gene, enterotoxin, and toxin genes were more prevalent. Different genes involved in biofilm production were detected with bap present only in animal-derived isolates and sasC present in both isolates, however, with a higher prevalence in the human-derived isolates. Biofilm capability was higher in human-derived isolates mainly associated to the sasC gene. Conclusions: The overall results indicate that human S. aureus isolates are more virulent and resistant than the isolates of animal origin randomly selected with no infection anamnesis. This study confirms that selection for more virulent and resistant S. aureus strains is related to the clinical practice.