Objective: Staphylococcus aureus is a significant pathogen associated with a wide range of infections, from mild skin conditions to severe systemic diseases. Its virulence is driven by various factors, including antibiotic resistance and biofilm formation, which complicate treatment and management. Understanding the genotypic and phenotypic characteristics of S. aureus is critical for addressing the rising prevalence of multidrug-resistant strains, particularly in Jordanian healthcare settings. Objective: This study aims to investigate the genotypic and phenotypic profiles of S. aureus isolates from diverse clinical samples in a Jordanian hospital. Methods: A total of 70 S. aureus isolates were collected from various clinical samples. Antibiotic susceptibility testing was performed using 15 antimicrobial agents. Biofilm formation was evaluated using Congo Red Agar (CRA) and Tissue Culture Plate (TCP) methods. The presence of the icaA gene and, recA gene a key biofilm-associated genetic marker, were assessed through PCR analysis. Results: High resistance rates were observed for Nalidixic acid (75.8%) and Erythromycin (75.7%), while Linezolid exhibited the highest sensitivity (89.8%). TCP and CRA methods identified 24 and 22 isolates, respectively, as strong biofilm producers. Genotypic analysis confirmed the icaA gene in 30 isolates, correlating with biofilm production. Older age groups (≥60 years) and females showed higher infection prevalence. Conclusions: This study highlights the alarming levels of antibiotic resistance and the critical role of biofilm production in S. aureus pathogenicity. The findings underscore the need for enhanced antimicrobial stewardship, targeted therapeutic strategies, and continuous surveillance to combat S. aureus-related infections effectively in Jordanian healthcare settings.