Background and Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important pathogens in hospitals. Hospital staffs can be one of the main sources of this organism in nosocomial infections. Identification and control of colonized health care workers can reduce the incidence of methicillin-resistant Staphylococcus aureus (MRSA). Mupirocin is a topical antibiotic and inhibits the activity of the most of grampositive Cocci. Mupirocin resistance emerges in a short time after taking this antibiotic. The main goal of this study is to evaluate the prevalence of asymptomatic carriers of methicillin- and mupirocin-resistant Staphylococcus aureus by Multiplex PCR method among Fasa Hospital Staffs, Fars Province, Iran. Materials and Methods: In this study, 150 samples of Staphylococcus aureus isolated from the hospital staffs were analyzed and confirmed by biochemical and microbial methods. Disk diffusion method was used to determine the level of strains susceptibilities toward methicillin and mupirocin. Then the presence of mec-A, mup, nuc genes was examined using Multiplex PCR. 150 people were included in this study, including 120 women and 30 men. Results: Of the 150 studied people, 53 cases including 42 women and 11 men were Staphylococcus aureus carriers. Four individuals among 11 studied men were resistant to methicillin and none of them was resistant to mupirocin. A number of 11 cases among 42 studied women were resistant to methicillin and 2 women were resistant to mupirocin. Conclusion: Prevalence of methicillin- and mupirocin-resistant Staphylococcus aureus among Fasa Hospital Staffs was moderate and relatively low. Permanent control of carriers and their treatment can prevent spread of the bacteria and those infections caused by them.