Staphylococcus aureus is considered an important human pathogen that produces a variety of exotoxins and develops resistance to several antibiotics. The present study was conducted to investigate various exotoxin and antimicrobial resistance genes of S. aureus isolates from patients referred to military hospitals in Tehran, using a multiplex PCR assay. Eighty clinical isolates of S. aureus were obtained from patients referred to three military hospitals in Tehran. A multiplex PCR assay was used for detection of enterotoxins A to D (sea, seb, sec, and sed,), toxic shock syndrome toxin 1 (tsst), and exfoliative toxins A and B (eta and etb), as well as antibiotic resistance genes, including mecA, aacA-aphD, erm(A), erm(C), tetK, tetM, vat(A), and vat(C). The frequency of MRSA strains was 63.7% (n = 51). The prevalence of sea, sec, seb, sed, tsst, and eta among S. aureus isolates was 51.2%, 23.7%, 15%, 3.7%, 33.7%, and 3.7%, respectively. None of the isolates possessed etb. The frequency of the resistance genes, was determined as follows: mecA (63.7%), erm(A) (33.7%), erm(C) (45.6%), tetK (33.7%), tetM (23.7%), aacA-aphD (21.2%), vat(A) (0%), and vat(C) (0%). Our findings indicated a high incidence of sea- and tsst- positive and MRSA strains with higher rates of antibiotic resistance.