Background: Urinary tract infections (UTIs) are among the most common infections and can cause numerous complications of the renal system. This study aimed to assess the prevalence of uropathogens and their antibiotic susceptibility patterns in Al-Madinah Al-Munawarah, Saudi Arabia. Methods: Data was collected from patients with UTIs presented at King Fahad General Hospital in Al-Madinah Al-Munawarah, Saudi Arabia. In this retrospective cross-sectional study, UTI microbial-causing agents and antimicrobial resistance profiles identified using automated systems, Phoenix and VITEK2, were collected between July 2022 and June 2023. In addition, minimal demographic data, including date of collection and sex and age of patients were collected and analyzed using Chi-square test. Results: The study included 1394 patients positive for UTI, comprising 50.57% males and 49.43% females (chi-square goodnessof-fit, p > 0.999). Microbial identification and antimicrobial susceptibility tests were performed on UTI-positive cultures. Among UTIs, mono-infection, caused by a single pathogen, was the most prevalent, accounting for 88.16% of cases, whereas poly-infection (caused by multiple pathogens) presented at 11.9%. The most prevalent UTIs' pathogens were E. coli (30.59%), followed by Klebsiella pneumoniae (21.40%), Enterococcus faecalis (8.46%), Pseudomonas aeruginosa (7.81%), Streptococcus agalactiae (6.35%), Enterococcus faecium (3.01%), Proteus mirabilis (3.01%), Enterobacter cloacae (2.52%), Candida sp. (2.44%), Acinetobacter calcoaceticus-baumannii (1.95%), Staphylococcus aureus (1.79%), and Enterobacter aerogenes (1.30%). The most dominant pathogens that coexisted with other uropathogens to cause UTIs were K. pneumoniae and P. mirabilis (9.32%, chi-square 5.550, p = 0.018), K. pneumoniae and P. aeruginosa (8.07%, chi-square 6.285, p = 0.012), K. pneumoniae and E. faecalis (7.45%, chi-square 5.785, p = 0.016), Candida sp. and Enterococcus faecium (4.97%, chi-square 9.176, p = 0.002, and Candida sp. and Acinetobacter calcoaceticus-baumannii (3.11%, chi-square 4.312, p=0.038)). Among the uropathogens, gram-negative pathogens showed resistance to most of the tested antimicrobials (ampicillins, cephalosporins, fluoroquinolones, trimethoprim-sulfamethoxazole, aztreonam, and nitrofurantoin). High rates of resistance were identified to cephalosporins, amoxicillin-clavulanic acid, and trimethoprim-sulfamethoxazole. Conclusion: This study reported UT mono-infection and poly-infection in Al-Madinah Al-Munawarah, Saudi Arabia, with a predominant representation from gram-negative bacteria, Enterobacteriaceae. Most of the UT microbial strains showed a highly resistant profile.