Background and Objectives: Urinary tract infection (UTI) is a common infection affects people of different ages. It is important to explore the antibiotics susceptibility of the bacterial agents to improve the empirical antibacterial prescription because of emerging of multi-drug resistant (MDR) bacteria. Materials and Methods: This is a retrospective observational study including 322 patients with UTI at the largest hospital at the center of Al-Basrah Governorate in the far south of Iraq from August 2018 to November 2019. Bacterial isolates from urine samples with significant bacteria were investigated by automated VITEK® 2 compact system to determine the causative bacteria and their antibiotics susceptibility. Results: Escherichia coli and Klebsiella pneumoniae were the first and second most frequent Gram-negative isolates, whereas Staphylococcus haemolyticus and Enterococcus faecalis were the first and second most frequent Gram-positive isolates. Fosfomycin, tigecycline, colistin, meropenem, imipenem, amikacin and nitrofurantoin had high susceptibility rates against Gram-negative isolates. Nitrofurantoin, tigecycline, daptomycin, teicoplanin, vancomycin and linezolid had a high effect against Gram-positive isolates. Conclusion: The leading causative isolates especially the most predominant Gram-negative isolates E. coli and K. pneumoniae show high resistance rates against important antibiotics including penicillin/β-lactamase inhibitors piperacillin/tazobactam, ceftazidime cefepime, ciprofloxacin, levofloxacin and trimethoprim/sulfamethoxazole which call for reconsidering them for treatment of UTI.