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Objective: Escherichia coli (E. coli) are gram-negative facultative anaerobes which are commonly found in the lower intestine. Biofilm production in E. coli promotes colonization and leads to an increase rate of infections, and such infections may be difficult to treat as they exhibit multidrug resistance (MDR). Methods: 50 strains of uropathogenic E. coli were collected from Clinical Microbiology laboratory at Saveetha medical college and hospital for a time period of 3 mo. Strains were identified by conventional biochemical methods. Biofilm formation and quorum sensing analysis were performed by the Microtitre plate method and Thin Layer Chromatography method (TLC), respectively. Results: In this study, 46 (92%) of E. coli strains were strong, 3(6%) were intermediate and 1(2%) were weak biofilm producers. From TLC analysis, 34 (68%) of the strains produced Acyl Homoserine Lactone molecules. Out of which, 16 isolates were shown unknown analytes of Retardation factor (Rf) value greater than 1. The Rf values identified were 3 unsubstituted C4 (5), 3 unsubstituted C6 (3), 3 oxo C8 (3), 3 oxo C4 (4), 3 oxo C6 (2), 3 oxo C1 (1). Conclusion: In this study, 100% of isolates were biofilm producers. Of which 18 strains produced known Acyl Homoserine Lactone molecules and 16 isolates produced unknown analytes. Thus, quorum sensing molecules plays a major role in biofilm formation.