Background: Healthcare-associated infections appear in a patient receiving medical attention at a hospital or other healthcare facility and develops infections that weren't present when they were admitted. One of the most important organisms causing infections acquired in hospitals is Staphylococcus aureus (S. aureus). For many bacterial species, Random Amplified Polymorphic DNA (RAPD) is a low-cost but effective typing technique. Objectives: To determine the genotypic polymorphism, the level of genetic relatedness, and the antimicrobial sensitivity and resistance among various S. aureus isolates. Methods: The following tests were performed on 50 samples that were obtained using strict aseptic precautions: sample culture, isolate identification using colony morphology, Gram stained film, biochemical responses, and antibiotic susceptibility testing. After RAPD-PCR, Syngene gene tool software was used to analyse the fingerprint pattern. Results: The most prevalent isolated organism was S. aureus. as it represented (70%) of isolated pathogens. Vancomycin and linezolid were effective against S. aureus isolates. The isolates of S. aureus were more common in nasal swab (42.9%) followed by wound (28.5%) then urine &endotracheal aspirate (14.3%). Patterns of RAPD-PCR of S.aureus isolates generated with primer GEN1-50-01 (5'GTGCAATGAG-3') resulting in several polymorphic bands and a dendrogram was created. Conclusions: S. aureus is an important cause of HCAI, Health care workers represent an important source of HCAI, RAPD –PCR technique was an easy and rapid one to perform with a good discriminatory power in typing (fingerprinting) of Staphylococcus aureus.