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<i>S. aureus</i> is a pathogenic bacterium that causesinfections. Its virulence is due to surface components, proteins, virulence genes, <i>SCCmec</i>, <i>pvl</i>, <i>agr</i>, and <i>SEs</i>, which are low molecular weight superantigens. <i>SEs</i> are usually encoded by mobile genetic elements, and horizontal gene transfer accounts for their widespread presence in <i>S. aureus</i>. This study analyzed the prevalence of MRSA and MSSA strains of <i>S. aureus</i> in two hospitals in Greece between 2020–2022 and their susceptibility to antibiotics. Specimens collected were tested using the VITEK 2 system and the PCR technique to detect <i>SCCmec</i> types, <i>agr</i> types, <i>pvl</i> genes, and <i>sem</i> and <i>seg</i> genes. Antibiotics from various classes were also tested. This study examined the prevalence and resistance of <i>S. aureus</i> strains in hospitals. It found a high prevalence of MRSA and that the MRSA strains were more resistant to antibiotics. The study also identified the genotypes of the <i>S. aureus</i> isolates and the associated antibiotic resistances. This highlights the need for continued surveillance and effective strategies to combat the spread of MRSA in hospitals. This study examined the prevalence of the <i>pvl</i> gene and its co-occurrence with other genes in <i>S. aureus</i> strains, as well as their antibiotic susceptibility. The results showed that 19.15% of the isolates were <i>pvl</i>-positive and 80.85% were <i>pvl</i>-negative. The <i>pvl</i> gene co-existed with other genes, such as the <i>agr</i> and enterotoxin genes. The results could inform treatment strategies for <i>S. aureus</i> infections.