Background and Objectives: Uropathogenic Escherichia coli (E. coli) (UPEC) accounts for 70-95% of community-acquired urinary tract infections (UTIs) and a significant proportion of nosocomial UTIs. This study aimed to characterize the phenotypic and genotypic characteristics of E. coli isolates from symptomatic UTI patients and evaluate their antimicrobial susceptibility patterns. Materials and Methods: A hospital-based observational study was conducted at Aarupadai Veedu Medical College and Hospital, Puducherry, India, from August 2022 to April 2024. A total of 106 UPEC isolates were obtained from symptomatic UTI patients. Antimicrobial susceptibility testing (AST) was performed using the Kirby-Bauer method, and virulence genes (hlyA, fimH, papC) were detected using PCR. Results: The mean age of patients was 49.7 years, with a female predominance (69.8%). Diabetes mellitus was the most common comorbidity (29.2%). Fever (60.4%) and dysuria (38.7%) were the most common symptoms. AST showed high susceptibility (>90%) to amikacin, nitrofurantoin, meropenem, and piperacillin/tazobactam, while >60% resistance was observed to cefotaxime and ceftazidime. Phenotypically, 30.2% of the isolates produced mannose-resistant hemagglutinins, and 17.9% produced hemolysin. ESBL production was found in 46.3%. Biofilm production was moderate in 65.1%, weak in 30.2% and strong in 4.7% and significantly correlated with multidrug resistance (p<0.05). Genotypically, 80.2% had fimH, 51.9% had papC and 20.8% had hlyA. papC was associated with reduced cefotaxime susceptibility (p<0.05). Conclusion: The study highlights the significance of phenotypic and genotypic characterization in understanding UPEC virulence and resistance patterns, and emphasizes the need for targeted empiric therapy to improve UTI management. © 2025 Elsevier B.V., All rights reserved.