The study isolated and characterized clinical and non-clinical S. aureus strains in Ile-Ife, Nigeria using phenotypic and molecular methods. Eight hundred and fifty samples of different cultures were taken from clinical and nonclinical sources. The clinical sources were the routine specimens of wound swabs, urine, stool, blood and sputum from the Department of Microbiology and Parasitology laboratory of the Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC) Ile-Ife. The non-clinical samples were obtained from the nasal cavity of apparently healthy food handlers at restaurants in Obafemi Awolowo University campus and food vendors in Ile-Ife central market. Samples were cultured on mannitol salt agar and incubated at 37°C for 24-48 hours. S. aureus were isolated and identified based on mannitol fermentation, Gram's reaction, positive results for catalase, coagulase and DNAse tests. Susceptibility of the isolates to nine different antibiotics was tested using the disk diffusion technique. Molecular detection of plasmid, mec A, nuc genes was carried out on representative isolates using polymerase chain reaction (PCR). The data generated were subjected to statistical analysis using T-Test. Of the 50 representative clinical and non-clinical isolates, 44% contained plasmid DNA with molecular weight ranging from 562 to 23,490 kb, while 18% had mec A and all selected representative isolates had the nuc gene. Only two (4%) out of the 50 S. aureus isolates investigated, were of PVL virulent strains attributed to urine sourced from both Pelvic inflammatory disease and wound of infected surgical implant individuals. These observations underscore the importance of confirming phenotypic identification of S. aureus by molecular techniques.