Background: Staphylococcus aureus ranks among the leading causes of serious nosocomial infections. One critical route for the spread of this bacterium within hospitals is via asymptomatic carriers, particularly healthcare providers. Staphylococcus aureus can exist as part of the normal skin flora and within the anterior nostrils of individuals, making healthcare providers a significant vector for transmission. Several genes associated with virulence, such as toxic shock syndrome toxin-1 (tsst-1), alpha-toxin (hla), and panton-valentine leucocidin (pvl), play pivotal roles in the pathogenicity and severity of infections caused by S. aureus. Objectives: This study aimed to characterize S. aureus nasal carriage among healthcare providers in an intensive care unit (ICU), with a particular focus on antibiotic resistance profiles and the prevalence of virulence genes. Methods: Nasal swabs were collected from 120 healthcare workers in the ICU of Ganjavian hospital, Dezful, Iran. Standard microbiological procedures were employed for S. aureus detection. Antibiotic susceptibility testing was conducted using both disk diffusion and minimum inhibitory concentration methods. Polymerase chain reaction (PCR) was employed to identify the presence of the mecA gene and the virulence genes hla, tsst-1, and pvl. A statistical analysis was performed to evaluate the data. Results: The study revealed that 12.5% of healthcare providers were carriers of S. aureus, with 26.6% of them harboring methicillin-resistant S. aureus (MRSA) strains. Antibiotic resistance patterns varied, with a notable resistance to erythromycin and penicillin. The hla gene was detected in 66.6% of S. aureus strains; nevertheless, the tsst-1 and pvl genes were not identified. The study suggests a potential association between high expression of the hla and mecA genes and antibiotic resistance. Conclusions: This study underscores the prevalence of S. aureus nasal carriage, antibiotic resistance patterns, and the distribution of virulence genes among healthcare providers in an ICU setting. The findings emphasize the significance of continuous surveillance and infection control strategies to mitigate the transmission of S. aureus and associated infections within healthcare facilities. The study recommends routine screening of ICU healthcare providers for asymptomatic S. aureus carriers and appropriate interventions to eliminate colonization.