The objective of this study was to describe the prevalence, microbiological profile, bacterial resistance, and the sensitivity to antibiotics of microorganisms causing urinary tract infection (UTI) at a single-site tertiary referral hospital in the western region of Mexico. Methods: A total of 5895 culture samples processed at the microbiology laboratory from August 1, 2014, to July 31, 2015, were analyzed. results: A total of 5895 samples for urine cultures (UC) were collected, of which 3363 were taken in women (57.05%) and 2532 in men (42.95%). A prevalence of 24% was calculated. From 1444 positive UC, 1512 microorganisms were isolated; the major etiological agent was Escherichia coli, representing 67.28% followed by Pseudomonas with 7.12%. With respect to fungi, Candida glabrata was found as the most common agent. Susceptibility to daptomycin and linezolid was 100%, and meropenem, 91.4%. Highest antimicrobial resistance was found for ampicillin (77.47%) and moxifloxacin (72.89%). Nearly 49% of E. coli strains and 27% of Klebsiella pneumoniae strains showed extended-spectrum beta-lactamase (ESBL) production. Conclusions: Bacterial UTI persists as one of the most common infections affecting all age groups and both genders. As in other countries, E. coli ranks first in Mexico, with 67.28%, and nearly 50% of the strains produce ESBL.