Staphylococcus aureus is an important cause of nosocomial and community associated infections all over the world. MRSA carriage is an important risk factor for subsequent infection, with the anterior nares, being the primary predilection site. Mupirocin is a topical narrow spectrum antibiotic that has been used for the treatment of different types of staphylococcal skin infections and has served as an important agent in the control of MRSA outbreaks because of its useful effect in the eradication of MRSA nasal carriage in patients and health care workers. Unfortunately mupirocin resistance in S. aureus has been reported with subsequent reduction in mupirocin effectiveness. Resistance to mupirocin is phenotypically divided into two groups, low-level resistance, with MICs of 8 to 256 µg/ml, and high-level resistance, with MICs of ≥512 µg/ml. The present study aimed at determining mupirocin resistance among MRSA isolates obtained from patients and health care workers, detecting the type of mupirocin resistance, whether low- or high-level resistance by the disc diffusion method and further confirmation by the E-test for only mupirocin-resistant isolates. Comparing the concordance of results of the mupirocin disc diffusion method and the E-test to determine whether the disc diffusion method could be a cheap accurate alternative to the E-test. This study was conducted on 100 MRSA isolates (50 isolated from different clinical samples from patients at Cairo University Hospitals and 50 isolated from nasal swabs obtained from healthcare workers during the period from August 2012 to July 2013. All S .aureus isolates obtained from clinical samples or carriers proved to be MRSA as they showed resistance to 30µg cefoxitin discs as evidenced by zone diameter ≤ 21mm. Mupirocin resistance was detected by disc diffusion method using (5, 200µg discs) then confirmed by the use of E-test. 5/50 (10%) of MRSA isolates obtained from patients showed mupirocin resistance and that 1/5(20%) with HL-MR and4/5(80%) with LL-MR, while 2/50(4%) of MRSA isolates obtained from health care workers showed mupirocin resistance and the 2(100%) isolates with HL-MR. It is important to eradicate MRSA colonization in both patients and health care workers to prevent its spread in the hospital or to the community. The rising emergence of mupirocin resistance and subsequent reduction in its effectiveness necessitate the need for other antibiotics to decolonize the nose in case of MRSA carriage.