Background: Staphylococcus aureus with concurrent resistance to antibacterial agents is emerging globally. This emergence might be due to the production of different virulence determinants, notably Panton-Valentine leukocidin (PVL). Objectives: This study aimed to investigate the genetic characteristics of PVL-positive S. aureus strains isolates from clinical samples. Methods: An epidemiological study was conducted on 65 S. aureus isolates carrying pvl genes. An antibiogram test by the disk diffusion and broth microdilution methods was conducted to assess antimicrobial resistance profiles. Results: All detected methicillin-resistant S. aureus (MRSA) isolates were confirmed by mecA polymerase chain reaction (PCR) assays. The PVL-positive isolates were characterized using multiplex PCR assay to detect staphylococcal cassette chromosome mec (SCCmec) and agr types. The PVL frequency was 19.5% and 17.6% in MRSA and methicillin-susceptible S. aureus (MSSA), respectively. Among the PVL-positive isolates, 66.2% and 33.8% were MRSA and MSSA, respectively. Multidrug resistance amounted to 84.6% of the isolates (MRSA: 61.5%, MSSA: 23.1%). Staphylococcal cassette chromosome mec III was dominated (55.8%; 24/43). The most commonly identified agr was type III (53.8%; 35/65). Resistance to vancomycin amounted to 12.3% of the isolates, and all belonged to agr type III and SCCmec type III. The frequency of inducible and constitutive clindamycin resistance among PVL-positive MRSA strains (12.3% and 26.1%) was higher than PVL-positive MSSA strains (7.7% and 15.4%). Most constitutive and inducible clindamycin resistance isolates belonged to agr type III (26.2% and 18.5%) and SCCmec type III (each 27.9%). In the present study, 32.3% of the isolates were confirmed as mupirocin resistant, and all were MRSA, 9 (42.9%) and 12 (57.1%) isolates of which exhibited high-level mupirocin resistant (HLMUPR) and low-level mupirocin resistant phenotypes. All HLMUPR MRSA isolates belonged to SCCmec III and recovered from wound samples. Conclusions: The emergence of vancomycin-resistant S. aureus strains among PVL-positive S. aureus strains in Iran is a serious alarm and seems to be becoming the greatest concern in the treatment of staphylococcal infections in the healthcare setting. The present study reinforces plausible direct transfers between community and nosocomial PVL-positive S. aureus types.