Introduction: Urinary tract infections (UTI) are one of the most common human bacterial infections encountered by physicians. The risk of resistant microbes is emerging as a result of the overuse of antibiotics treatments. The presence of pathogens with increased resistance to antimicrobial agents makes UTIs difficult to treat. This study was aimed at determining the prevalence of the pathogens that cause UTIs, as well as the antibiotic susceptibility of these isolates. Materials and methods: This prospective study was conducted from February 2020 to April 2020; a total number of 200 urine samples were collected from patients who daily attended TMC Libya. Bacterial pathogens were determined by bacteriological culture methods and Antimicrobial susceptibility testing was done by using the disc diffusion method. Results: Out of 200 samples, 110 cases had a positive culture. The dominant bacterial pathogens were Gram-negative that being with Escherichia coli (49, 55.68%), followed by Klebsiella pneumonia (18, 20.46%), Pseudomona aeruginosa (9, 10.23%), Proteus mirabilis (8, 9.09%), Enterobacter aerogenes (2, 2.27%), Citrobacter freundii (2, 2.27%). Gram-positive bacteria were Staphylococcus aureus 20 (90.91%) followed by S. saprophyticus (2, 9.01%) of the isolate’s strains. The isolated uropathogen showed increased levels of resistance to antibiotics. Where the Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus indicated the highest antibiotic resistance to Nitrofurantoin, Sulfamethoxazole/trimethoprim, Tetracycline, Ciprofloxacin, Metronidazole and also revealed the most sensitivity to Cefixime followed by doxycycline and ceftriaxone. Conclusions: The obtained results emphasized the emergence of highly resistant bacteria to most of the tested antimicrobials and propose the need for physicians to change their treatment pattern depending on antimicrobial susceptibility results.