Escherichia coli (E. coli) is responsible for 70%-95% of urinary tract infections (UTIs), with biofilm formation significantly contributing to antibiotic resistance. This study investigates the relationship between biofilm formation and antibiotic resistance in uropathogenic E. coli, and assesses the effect of Ciprofloxacin (CIP) on biofilm development. E. coli strains were isolated from UTI patients and their biofilm-forming abilities were evaluated using Tube and Microtiter plate assays. The minimum inhibitory concentration (sub-MIC) of CIP was determined through resazurin assays and growth curve analysis. The impact of CIP on biofilm formation was assessed by comparing biofilm production in treated versus untreated isolates, with statistical significance determined using the t-test (p < 0.05). Out of 35 samples, 30 E. coli isolates were identified, with 98% exhibiting biofilm production of varying intensities (strong, moderate, and weak). The sub-MIC of CIP was found to be 0.1 mg/ml, which reduced biofilm formation by 75%, lowering the mean biofilm production from 0.06 to 0.02, with statistically significant results (p < 0.05). The findings suggest a notable link between biofilm formation, antibiotic resistance, and E. coli pathogenicity. Sub-MIC CIP effectively inhibits biofilm formation without adversely affecting bacterial growth, indicating its potential as a therapeutic option for managing biofilm-associated bacterial infections.