Background Urinary tract infections (UTIs) are among the most common bacterial infections globally, with Enterobacteriaceae, particularly and , being the primary causative agents. The rising prevalence of antimicrobial resistance (AMR) in these pathogens has complicated treatment, posing a significant public health challenge. In resource-limited settings like Southwestern Uganda, limited surveillance data and inappropriate antimicrobial use exacerbate the AMR crisis. Understanding the antibiotic susceptibility profiles of uropathogenic Enterobacteriaceae is crucial to inform empirical treatment and guide local antimicrobial stewardship efforts. This study investigated the antimicrobial susceptibility patterns of Enterobacteriaceae isolated from UTI patients to commonly used antibiotics. Methods A hospital-based cross-sectional study was carried out in Bwizibwera Health Center IV and Rubaya Health Center III in rural Southwestern Uganda, targeting 455 individuals who were confirmed to have Enterobacteriaceae infections. These participants were drawn from a pool of 2,371 patients presenting with urinary tract infections (UTIs) at the two health centers. Identification of Enterobacteriaceae was performed using a range of biochemical tests, and antimicrobial susceptibility patterns were assessed using the Kirby-Bauer disk diffusion technique with specific antibiotics. The data were analyzed and summarized as frequencies and percentages, with results presented in tables and graphs. Results Out of the 2371 participants, 455 (19.2%, CI 17.6 - 20.8) tested positive for uropathogenic Enterobacteriaceae. Out of the 455, the majority were females (398/455, 87.5%). The mean age of the study participants was 41.6 (SD 19.7) years. Each of the 455 study participants had a single uropathogen. Organisms were highly resistant to tetracycline with a proportion of 89.5% (407/455) and least resistant to meropenem 76 out of 455 (16.7%) and ceftriaxone 79 out of 455 (17.9%). Multidrug resistance (MDR) was observed in 114 out of 455 (25.05%) of the organisms, whereas 10.77% (49/455) of the organisms exhibited pan-drug resistance (PDR). In addition, MDR was highest in isolates from females, and MDR and PDR were highest among individuals 60 years and above. Conclusion Our study revealed a high prevalence of Enterobacteriaceae uropathogens isolated in rural South Western Uganda predominantly affecting women. The organisms showed the highest resistance to tetracycline and varying degrees of antibiotic resistance (MDR, extensive drug resistance (XDR), and PDR), including carbapenem-resistant strains of , , and spp., posing a significant public health threat. These findings highlight the urgent need for revised antibiotic prescription practices, strengthened antibiotic stewardship, and continuous surveillance of uropathogen susceptibility patterns to guide effective empirical treatment strategies.