Background: Staphylococcus aureus (S. aureus) is a well recognized nosocomial and community acquired pathogen which is implicated in causing a wide spectrum of superficial, deep pyogenic infections and toxin mediated illnesses. Localized infections sometimes progress to systemic infections, while ‘spontaneous’ bacteraemia also occur without an evident septic focus, particularly in chronic debilitated immunocompromised patients. Emergence of drug resistance to penicillins and penicillinase-resistant penicillins (i.e., oxacillin and methicillin) is a globally recognized problem. Methicillin Resistant Staphylococcus aureus isolates are also important with respect to the range of nosocomial infections it causes, leading to an increase in the hospital expenditure and mortality or morbidity rate. The increased prevalence of such resistant strains has narrowed down the list of available therapeutic options. Therefore, information regarding the prevalence of S. aureus infections in a health care setting and determining its current antibiotic resistance profile becomes crucial in selecting appropriate treatment regime. Therefore, the current study was done in the department of Microbiology to determine the prevalence of S. aureus infections and the antibiotic resistance pattern of S. aureus isolates from various clinical specimens at our tertiary care hospital in North India. Materials and Methods: A one year prospective study was carried out in the Department of Microbiology, at a tertiary care hospital in North India where non-duplicate strains of S. aureus isolated from various clinical specimens received in the Microbiology laboratory were studied. All S. aureus isolates were subjected to Antibiotic Susceptibility Testing using Kirby Bauer’s disk diffusion method on Mueller Hinton Agar plates in accordance to CLSI guidelines. The antibiotics tested included Penicillin (10U), Amoxicillin-clavulanic acid (20/10μg), Sulphamethoxazole-trimethoprim (1.25/23.75μg), Ciprofloxacin (5μg), Erythromycin (15μg), Clindamycin (2μg), Vancomycin (30μg), Teicoplanin (30μg) and Linezolid (30μg). Results: A total of 23,699 clinical specimens were processed in the laboratory while conducting this study, from which 1233 clinical isolates of S. aureus were identified and processed further. Among all clinical specimens, pyogenic samples (63.1%) yielded maximum number of S. aureus strains followed by blood samples (29.9%) and urine samples (4.8%). S. aureus infection was more evident in hospitalized 71.2% patients than in OPD patients 28.8%. Seasonal variation was also seen in isolation of S. aureus, with a higher percentage of isolates obtained during summer season than during winter season. On antibiotic susceptibility testing, 49.6% strains were Methicillin Resistant. Majority of the isolates were found resistant to Penicillin (92.1%), followed by Erythromycin (59%). Almost half of the total isolates were resistant to Sulphamethoxazole-Trimethoprim (49.3%) followed by Amoxicillin- Clavulanic acid (47.8%), Ciprofloxacin (43.4%) and Clindamycin (18.4%). Antibiotics to which all isolates showed 100% susceptibility included Vancomycin, Teicoplanin, Linezolid. Conclusion: Given the high prevalence of resistance to antibiotics seen in this study, effective treatment of infections caused by multidrug resistant Staphylococcal strains may become challenging. Drugs like Vancomycin, Teicoplanin and Linezolid promise to work as miracle drugs against the multidrug resistant MRSA strains but we need to warrant judicious use of these wonder drugs to conserve them for future use.