Background: Staphylococcus aureus is the most common cause of nosocomial and community acquired infections. The infections caused by the drug resistant strains of Staphylococcus aureus are difficult to treat in hospital settings. Objective: This study was aimed to determine antimicrobial susceptibility patterns of Staphylococcus aureus at the University of Gondar Teaching and Referral Hospital, Northwest Ethiopia. Methods: A retrospective cross sectional study was conducted from September 2013 to February 2014 to investigate S. aureus prevalence and its antibiotic susceptibility patterns among different specimens. Antibacterial susceptibility testing was done by disk diffusion method on Mueller-Hinton agar. ens collected from various sections of the Hospital in the University. Completeness of the collected data was checked and entered into the computer. Chi-square test was used to check whether there is the association between the socio-demographic characteristics of the study participants and positivity to S. aureus. P-value less than 0.05 was considered as statistically significant. Results: Of the 4321 different samples cultured, 309 of them were positive for S. aureus. The overall prevalence of S. aureus was 7.2% and the prevalence in the type of samples cultured were: abscess (22%), followed by body discharges (19.2%), wound secretions (17.9%), pus (17.5%), blood (9.1%), urine (4.4%) and body fluid (0.6%). The sensitivity rates of the isolates from the highest to the lowest were: vancomycin (99.6%), cefoxitin (92.6%), clindamycin (89.5%), Ceftriaxone (86.7%), ciprophloxacillin (81.2%), gentamycin (80%), chloramphelicol (78%), norfloxicillin (65%), erythromycin (53.2%), co-trimoxazole (39.7%), penicillin (37.7%), ampicillin (36.3%), amoxicillin (34.5%) & tetracycline (30.6). One hundred and sixty six (53.7%) of the isolates showed multi-resistance to antimicrobial agents. Conclusion: Staphylococcus aureus isolates in this study showed higher multi-drug resistance patterns to several antimicrobials and thus further studies should be conducted in the hospital.