Staphylococcus aureus is opportunistic human pathogen that causes a variety of diseases. Out of 580 clinical specimens, 100 Staphylococcus aureus isolates were isolated and identified depending on cultural, morphological and different biochemical tests, in addition to molecular identification by using PCR with specific primer 16S rRNA. For biofilm detection, method of polystyrene microtitre plate was used and the results showed that 61% were biofilm producer and 39% were non-biofilm producer isolates. The different methods were applied for detection of methicillin resistant Staphylococcus aureus isolates and the identification of mecA gene by PCR was considered as the gold standard method comparing with other four phenotypic tests in sensitivity, specificity, positive predictive value, negative predictive value and accuracy of method. The results of cefoxitin showed that 50 (81.97%) were similar to mecA gene PCR 50 (81.97%) and sensitivity, specificity, positive predictive value, negative predictive value and accuracy of these test were 100%, while, the method of oxacillin disk diffusion, oxacillin agar screening and MIC for oxacillin showed less specificity, positive predictive value and accuracy. Whereas, sensitivity and negative predictive value for all methods equal to 100%. The results of cefoxitin disk diffusion test showed the highest specificity, accuracy compared to other phenotypic tests that were low precision in the determination of methicillin resistant Staphylococcus aureus isolates. On the other hand, It was found that there was a relation between femA and mecA genes, while no relation was observed between femB, mecC and with mecA.