Urinary tract infections are among the most common bacterial infections in clinical practice, and increasing antimicrobial resistance among uropathogens poses a serious challenge to empirical therapy, especially in tertiary care settings. In this study, we analyzed culture positivity rate, bacteriological profile, and distribution of urine isolates by patient location in a tertiary care hospital. This retrospective laboratory-based study was conducted in the Department of Microbiology of a tertiary care teaching hospital between January 2024 and December 2024. Urine samples were processed using standard semiquantitative culture techniques on cystine lactose electrolyte-deficient medium. Organism identification and antimicrobial susceptibility testing were performed using an automated BD Phoenix™ system. Extended-spectrum β-lactamase (ESBL) and carbapenem-resistance detection were interpreted according to Clinical and Laboratory Standards Institute guidelines. Of 3434 urine samples processed, 857 (24.9%) showed significant bacterial growth. Gram-negative bacilli predominated (77.4%), whereas Gram-positive cocci accounted for 22.6%. Culture positivity rate was higher among inpatients than outpatients. Among Gram-negative isolates, ESBL production was most common in Escherichia coli (63.4%; 312/492), and Klebsiella spp. (24.4%; 120/492). Carbapenem-resistant isolates included E. coli (47.4%, 81/171), Klebsiella spp. (28.1%, 48/171), Pseudomonas aeruginosa (14.0%, 24/171), and Acinetobacter spp. (7.0%, 12/171). ESBL producers demonstrated high resistance to cephalosporins and fluoroquinolones, with better susceptibility to nitrofurantoin, amikacin, and meropenem. Colistin retained the highest activity against carbapenem-resistant isolates (81.9%, 140/171). Gram-negative bacilli remain the predominant uropathogens in tertiary care settings, with an increasing burden of ESBL and carbapenem-resistance These findings underscore the need for continuous resistance surveillance, culture-guided therapy, and robust antimicrobial stewardship programs.