Background: Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) is a significant health concern, exacerbated by the production of extended-spectrum beta-lactamases (ESBLs), biofilm formation, and cell surface hydrophobicity. These traits contribute to antibiotic resistance and persistent infections. This study aimed to assess the prevalence of ESBL-producing UPEC isolates and their associations with biofilm formation, cell surface hydrophobicity, and antimicrobial resistance patterns in a tertiary care hospital in Imphal, India. Methods: This observational cross-sectional study was conducted from October 2022 to July 2024, analyzing 398 UPEC isolates. Biofilm production was evaluated using the Congo red agar and tissue culture plate methods, while cell surface hydrophobicity was assessed via the salt aggregation test. ESBL production was determined using the potentiated disc diffusion method, and antibiotic susceptibility testing was performed following CLSI 2022 guidelines. Statistical analyses were conducted using SPSS software, with significance set at p-value<0.05. Results: ESBL production was observed in 68.8% of isolates, and biofilm formation in 52.8%. Among ESBL producers, 58% demonstrated biofilm formation, significantly higher than non-ESBL producers (44%, p=0.009). Cell surface hydrophobicity was detected in 31.7% of isolates and was significantly associated with biofilm formation (p=0.000). However, no association was found between ESBL production and hydrophobicity (p=0.900). Biofilm-producing isolates exhibited higher resistance rates, particularly to third-generation cephalosporins and ciprofloxacin, while maintaining sensitivity to gentamicin, fosfomycin, and amikacin. Conclusion: This study highlights the high prevalence of ESBL-producing UPEC isolates and their enhanced biofilm-forming capacity, emphasizing the need for stringent antimicrobial stewardship and infection control measures.