Background: Staphylococcus aureus, particularly MRSA, poses significant challenges due to antibiotic resistance and multi-drug-resistant strains, complicating treatment in high-risk environments like ICUs. Objective: The study aimed to identify MRSA molecular types, assess prevalence among healthcare workers and patients, evaluate PVL gene presence, and evaluate infection control protocols' effectiveness in ICUs at Menoufia University Hospitals. Methods: A cross-sectional study was conducted from May 2022 to August 2023, involving 180 ICU patients and 69 HCWs. Clinical specimens were analyzed using microbiological and molecular techniques, including PCR for mecA and PVL gene detection, SCCmec typing, and PCR-RFLP of the coagulase gene. Antimicrobial susceptibility testing was performed using the Vitek-2 Compact system. Infection control compliance was evaluated through observational studies and risk assessments. Results: Results revealed 81 Staphylococcus aureus isolates (63 MRSA and 18 MSSA), with 17.5% of MRSA and 5.6% of MSSA strains harboring PVL genes. The Vitek-2 Compact system demonstrated high diagnostic accuracy (98.4% sensitivity, 94.4% specificity). MRSA strains exhibited high resistance to benzylpenicillin and fusidic acid but remained susceptible to tigecycline, linezolid, and teicoplanin. SCCmec typing showed that hospital-acquired MRSA (HA-MRSA) strains were mostly types I, II, and III, while community-acquired MRSA (CA-MRSA) strains were mainly types IV and V. CA-MRSA strains had a high PVL-positivity rate (71.4%), indicating increased virulence. Improved hand hygiene and personal protective equipment compliance were associated with reduced MRSA infection rates. conclusion: Continuous surveillance and strict infection control measures are crucial for addressing MRSA prevalence, with Vitek-2 Compact system recommended for accurate detection and PVL screening.