Objective:To detect in vitro biofilm formation of uropathogenic Escherichia coli(E.coli)(UPEC) strains isolated from urine specimens and also to determine their antimicrobial susceptibility pattern using 13 commonly used antibiotics.Methods:The present study comprised of 166 urine specimens collected from tertiary care hospitals in and around Coimbatore.South India. All the specimens were subjected to gram staining,bacterial culture and the E.coli strains were screened for biofilm formation using Tube Method(TM),Congo Red Agar(CRA) and Tissue Culture Plate method(TCP) respectively.Subsequently,the antimicrobial susceptibility test was performed by Kirby Bauer-disk diffusion method for the biofilm and non-biofilm producing E. coli strains.Results:Of the 100(60.2%) E.coli strains,72 strains displayed a biofilm positive phenotype under the optimized conditions in the Tube Method and the strains were classified as highly positive(17,23.6%),moderate positive(19.26.3%) and weakly positive(36.50.0%). similarly under the optimized conditions on Congo Red agar medium,biofilm positive phenotype strains were classified as highly positive(23,23%).moderate positive(37.37%) and weakly positive(40,40%).While in TCP method,the biofilm positive phenotype strains were also classified as highly positive(6.6%),moderate positive(80.80%) and weakly positive(14,14%),it didn’t not correlate well with the tube method for detecting biofilm formation in E.coli.The rates of antibiotic resistance of biofilm producing E.coli were found to be 100%for chloramphenicol and amoxyclav(amoxicillin and clavulanic acid),86%for gentamicin and cefotaxime.84%for ceftazidime,83%for cotrimoxazole and piperacillin/tazobactam,75%for tetracycline and 70% for amikacin,Conclusions:This study reveals the prevalence and antimicrobial susceptibility pattern of biofilm and non-biofilm producing uropathogenic E.coli strains. Objective: To detect in vitro biofilm formation of uropathogenic Escherichia coli (E.coli) (UPEC) strains isolated from urine specimens and also determine their antimicrobial susceptibility pattern using 13 commonly used antibiotics. Methods: The present study comprised of 166 urineites collected from tertiary care hospitals in and around Coimbatore. South India. All the specimens were subjected to gram staining, bacterial culture and the E. coli strain were screened for biofilm formation using Tube Method (TM), Congo Red Agar (CRA) and Tissue Culture Plate method (TCP) respectively. Published, the antimicrobial susceptibility test was performed by Kirby Bauer-disk diffusion method for the biofilm and non-biofilm producing E. coli strains. Results: Of the 100 (60.2%) E. coli strain, 72 The results of a biofilm positive phenotype under the optimized conditions in the Tube Method and the strain were classified as highly positive (17,23.6%), moderate positive (19.26.3%) and weakly positive (36 .50.0%). Similarly under the optimized conditions on Congo Red agar medium, biofilm positive phenotype strains were classified as highly positive (23,23%). Moderate positive (37.37%) and weakly positive (40,40%). While in TCP method, the biofilm positive phenotype was also classified as highly positive (6.6%), moderate positive (80.80%) and weakly positive (14,14%), it did not not correlate well with the tube method for detecting biofilm formation in E. coli. The rates of antibiotic resistance of biofilm producing E. coli were found to be 100% for chloramphenicol and amoxyclav (amoxicillin and clavulanic acid), 86% for gentamicin and cefotaxime. 84% for ceftazidime, 83% for cotrimoxazole and piperacillin / tazobactam, 75% for tetracycline and 70% for amikacin, Conclusions: This study reveals the prevalence and antimicrobial susceptibility pattern of biofilm and non-biofilm producing uropathogenic E. coli strains.