Background <p>Nowadays, increasing extended-spectrum beta-lactamase-producing Enterobacteriaceae, which were a common cause of urinary tract infection, have become a global concern because they induce resistance to most of the antimicrobial classes and make treatment difficult.</p> Objective <p>This study aimed to determine the prevalence of extended spectrum beta-lactamase producing Enterobacteriaceae, antibiotic susceptibility and its associated factors among patients suspected with urinary tract infection at Wachemo University Nigist Eleni Mohammed memorial Comprehensive Specialized Hospital, Hosanna, Ethiopia.</p> Methods <p>A hospital-based cross-sectional study was conducted from May 10 to July 31, 2024, involving 287 patients suspected of having urinary tract infections. Socio-demographic information, clinical data, and potential risk factors were collected using the Kobo Toolbox software. Urine samples were obtained from each participant, mixed thoroughly, and aseptically inoculated onto MacConkey agar and CLED agar using a standard calibrated loop. Identification of Enterobacteriaceae isolates was performed using standard bacteriological methods. Antibiotic susceptibility testing was carried out using the Kirby–Bauer disk diffusion technique. Extended spectrum beta-lactamase (ESBL) production was confirmed through the double-disk synergy test and other standard phenotypic methods. Data were entered and analyzed using SPSS version 29. Both univariate and multivariable analyses were conducted to assess associations between ESBL production and the collected variables.</p> Results <p>The proportion of urinary tract infections caused by Enterobacteriaceae was 33% (95% CI: 27.7–38.9). A total of 95 Enterobacteriaceae isolates were identified, with <i>Escherichia coli</i> predominating (62.1%), followed by <i>Klebsiella</i> spp. (23.2%). The overall prevalence of extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae was 26.3% (95% CI: 17.8–36.4), with <i>E. coli</i> accounting for 64% of cases and <i>Klebsiella</i> spp. for 24%. ESBL-producing isolates demonstrated complete resistance (100%) to cefotaxime and ceftriaxone. Resistance rates to other antibiotics were as follows: norfloxacin (80%), nalidixic acid (68%), ciprofloxacin (60%), amoxicillin-clavulanic acid (48%), gentamicin (36%), and amikacin (28%). In contrast, lower resistance was observed to meropenem (8%) and nitrofurantoin (20%). Multivariable logistic regression analysis identified three independent risk factors for ESBL production: a previous history of urinary tract infection (AOR = 8.002), prior urinary catheterization (AOR = 3.385), and history of urinary tract surgery (AOR = 4.535).</p> Conclusion <p>ESBL-producing Enterobacteriaceae pose a major challenge in urinary tract infections due to their high prevalence and resistance to commonly prescribed antibiotics. Prior urinary tract infections, catheterization, and urinary tract surgery were identified as independent risk factors, underscoring the importance of preventive strategies. These findings highlight the urgent need for region-specific antimicrobial stewardship programs, improved diagnostic capacity, and continuous surveillance to curb multidrug resistance and ensure effective patient outcomes.</p> Clinical trial number <p>Not applicable.</p>