Urinary tract infections (UTIs) are one of the commonest infections encountered by clinicians despite the widespread availability of antimicrobial agents. Such Infections caused by different bacterial pathogens that can be acquired through both hospitals and the community. The present study is aimed to isolation and diagnosis of Staph. aureus , detecting the optimal antimicrobial for the treatment of these infections. A total of (600) mid-stream urine samples were collected during the period from August to December, 2018 from patients who were complained from UTIs at AL-Hussain Teaching Hospital in AL-Nasiriyah City, Southern Iraq. The study included isolation and diagnosis of Staph. aureus based on morphological, microscopic characterization, biochemical tests and confirmed by API-20 and Vitek2 systems. In addition , all Staph. aureus isolates were subjected to the serological diagnosis of protein A by using a latex agglutination test and convention PCR technique was used to detect the presence of the 16S rRNA gene (353bp). 380 (63.33%) were positive isolates for bacteriological examination, the Staph. aureus was identified with 50 samples (13.5 %). In addition, all Staph. aureus isolates were assayed for antimicrobial susceptibility against 14 selected antibiotic discs by using the disc diffusion method. All isolates were completely resistant to Penicillin (P), Oxacillin (Ox), and Ampicillin (Amp). While the most effective antibiotics were Nitrofurantoin, Gentamycin, isolates were susceptible to these antibiotics in 76%, and 60%, respectively. Also, In the analysis of the nucleotides sequence of the partial 16S rRNA gene, the results were showed that these bacteria actually related to Staph. aureus ,according to current result, three of these isolates registered globally in the NCBI Gen bank, and the accession numbers of these isolates are (MK910079.1),( MK910080.1), and (MK910081.1).