Objectives To investigate the prevalence of urinary tract infection among patients at Messalata Central Hospital, Libya, to identify the causative bacteria, and to explore their resistance pattern to antimicrobials. Methods A total number of 1 153 urine samples were collected from patients, who attended daily to Messalata Central Hospital, Libya, in a study extended for one year. Antimicrobial susceptibility testing and isolates typing were done using Phoenix BD (BD diagnostic). Resistance was confirmed manually using agar disk diffusion method. Results Of the 1 153 urine samples tested, 160 (13.9%) samples were positive, from which 17 different, solely Gram negative, uropathogens were identified. Escherichia coli were the most prevalent (55.6%) bacteria, followed by Klebsiella pneumoniae subspecies pneumoniae (16.3%), Proteus mirabilis (6.3%), Pseudomonas aeruginosa (5.6%), Enterobacter cloacae and Klebsiella oxytoca (2.5%, each), Citrobacter koseri and Providencia rettgeri (1.9%, each), Acinetobacter baumannii, Enterobacter aerogenes and Proteus vulgaris (1.3%, each), and Aeromonas caviae, Citrobacter freundii, Cronobacter sakazakii, Enterobacter amnigenus biogroup 2, Pseudomonas putida and Serratia marcescens (0.6%, each). The isolated uropathogens showed increased levels of resistance ranged from 10.5% to 64.5%, with an overall resistance of 28.9%. Amikacin was the most effective antimicrobial followed by Imipenem and Meropenem (0%, 0.6% and 2.5% resistance, respectively); while, Cephalothin and Ampicillin were the least (80.6% and 90.0% resistance, respectively) effective. Conclusions The obtained results emphasized the emergence of highly resistant bacteria to most of tested antimicrobials and raise the alarm for physicians to change their treatment pattern depending on antimicrobial susceptibility results.