Background: Urinary tract infection (UTI) is a clinical condition with characteristic symptoms, colonization and multiplication of bacteria in significant numbers. At this juncture, antimicrobial resistance in uropathogens has become one of the major concerns globally. Therefore, the present study aimed to demonstrate the biofilm formation in uropathogenic Escherichia coli strains; characterize the phenotypic & genotypic virulence factors and their relationship with antimicrobial resistance. Methods and materials: In this study, 1000 suspected UTI cases were included. Urine samples were processed for culture and antimicrobial drug susceptibility testing. Only Escherichia coli isolates were further studied for production of biofilm, ESBL and haemolysin enzymes by phenotypic methods. Virulence genes, papEF, traT & PAI were detected by multiplex PCR. Molecular confirmation & characterization of virulence genes was done by Sanger sequencing. Results: From the 1000 urine samples, 395 E. coli were isolated. Higher resistance was observed with antibiotics - ampicillin (82.53%), cefuroxime (72.41%), amoxicillin-clavulanic acid (71.90%), ceftriaxone (66.58%), ciprofloxacin (65.82%) and cefepime (57.47%). In-vitro biofilm was formed by 71.39% isolates. Biofilm-forming strains developed higher resistance towards ampicillin, cefuroxime, amoxicillin-clavulanic acid, ciprofloxacin, cefepime and ceftriaxone. Phenotypic methods detected 62.3% isolates as ESBL producers and 40.2% were β-haemolytic. Virulence gene characterization revealed presence of traT in 73.2% strains, PAI & papEF among 62.9% and 33.5% strains. ESBL & Hemolysin producing UPEC exhibited higher resistant to ampicillin, amoxicillin-clavulanic acid, aztreonam, cefriaxone, cefuroxime, cefepime, ciprofloxacin, chloramphenicol & gentamicin. Statistically proved association was seen with gene RPAI and traT with antibiotics nitrofurantoin and amikacin. All the isolates were sensitive to nitrofurantoin, piperacillin-tazobactam, and imipenem. Strains showed 99% sequence identity and no mutations were detected. Conclusion: Increase in trend of resistance was observed with antibiotics which were routinely used to treat UTI. Results indicated significant correlation between phenotypic and genotypic virulence factors and antibiotic resistance. Antibiotics nitrofurantoin, piperacillin - tazobactam, and imipenem can be effective for severe UTIs. This study concludes that expression of virulence factors by UPEC strains is responsible for increased antibiotic resistance. Hence, characterizing the UPEC strains help clinicians to reach a better therapeutic outcomes and treatment regimens in this region.