Background: Staphylococcus aureus has developed antibiotic resistance, a global challenge that makes managing infections difficult. The accessory gene regulator (agr) is a quorum-sensing system virulence regulator. Objectives: The objective of this study was to investigate the relations between agr types, antimicrobial resistance, and biofilm formation among S. aureus isolates. Methodology: Various clinical samples from patients with nosocomial infections in ICUs of Tanta University Hospitals were included. S. aureus isolates were identified, antibiotic sensitivity testing was done using Kirby-Bauer disk diffusion method, mecA gene screening by PCR, the tissue culture plate technique was utilized to evaluate the biofilm formation, and agr typing was detected by multiplex PCR. Results: Seventy-six S. aureus isolates were obtained; they were highly resistant to ampicillin (90.8%), imipenem (73.7%) and no resistance for vancomycin or linezolid in all isolates. 56 isolates (73.68%) were MRSA while 20 isolates (26.31%) were MSSA. There is a significant variation in biofilm formation between MSSA and MRSA isolates. Both biofilm-forming and non-forming isolates exhibited a rise in antibiotic resistance. Among all isolates, agr I was the commonest type (61.8%) followed by agr III (18.4%). The existence of agr I was linked to biofilm development. Isolates with agr I were resistant to Erythromycin, Gentamycin, Ciprofloxacin, Levofloxacin, and Tetracycline among MRSA isolates. Conclusion: High rate of antibiotic resistance, and biofilm production among agr-positive S. aureus isolates were detected in ICUs. Therefore, the development of a new effective anti-virulence agent is promising for the treatment of S. aureus infection.