Introduction: Staphylococcus aureus is one of the most common causes of healthcare and communityassociated infections. Its remarkable ability to acquire antimicrobial resistance mechanisms and advantageous pathogenic determinants has contributed to emergence of infections in both nosocomial and community settings. Objective: To determine prevalence of Staphylococcus aureus and antibacterial susceptibility patterns in patients, nasal carriage of health personnel and objects of Dessie Referral Hospital. Methods: Cross sectional study was conducted at Dessie Referral Hospital from February 01 to May 30, 2013. Using a convenient sampling technique, 180 specimens of pus, blood, nasal swab and swab from hospital objects were collected and cultured by standard procedure. Growth identification was based on colony morphology, Gram staining and results of biochemical tests. Antibacterial susceptibility testing was done by disk diffusion method on Mueller-Hinton agar. Result: Overall prevalence of Staphylococcus aureus was 40.5% and its occurrence in inpatients, health personnel and objects was 57.5%, 40% and 34.3% respectively. Penicillin G (90.4%), nalidixic acid (93.2%), and amoxicillin (82.9%) showed high level of resistance, whereas, gentamicin (84.3%)), tetracycline (62.9%) chloramphenicol (63.6%), ciprofloxacin (61.6%), and kanamycin (64.4%) were relatively effective against Staphylococcus aureus infection. Vancomycin exhibited 100% susceptible in all study subjects. Conclusion: Staphylococcus aureus is still the most common cause of nosocomial infection and multi-resistant was very high and most of the isolates showed high levels of resistance to commonly used antimicrobials. In the absence of diagnostic bacteriologic services, vancomycin and gentamicin are the best therapeutic options to treat S. aureus infections.