Panton-Valentine Leukocidin (PVL) is a cytotoxin produced by Staphylococcus aureus that causes leukocyte destruction and tissue necrosis. Therefore, this study aimed to detect the rate, antimicrobial susceptibility, associated risk factors, and the phylogenetic relationship of PVL-positive S. aureus nasal carriers among patients and nurses. The research methods included the collection of 315 nasal specimens obtained from inpatients and nurses. The identification of S. aureus was confirmed by a coagulase test. The multiplex polymerase chain reaction technique was used to affirm PVL-positive S. aureus. The antibiotic sensitivity of S. aureus isolates was carried out using the disk diffusion method. The phylogenetic similarity of PVL-positive S. aureus was identified by pulsed-field gel electrophoresis. This study revealed that 160 out of 315 (50.8%) isolates were S. aureus. In addition, 7/160 (4.4%) had the lukS gene (six MSSA and one MRSA). The PVL-positive S. aureus isolates were 100% sensitive to gentamicin, linezolid, mupirocin, rifampin, trimethoprim-sulfamethoxazole, teicoplanin, tigecycline, and vancomycin. The risk factor analysis revealed that a longer hospital stay, nasogastric intubation, and runny nose were significant risk factors for patients to be PVL-positive S. aureus nasal carriers. A phylogenetic similarity analysis of PVL-positive isolates showed five models and they were distantly correlated. Therefore, the current study will provide knowledge to the hospital infectious control authority.