BACKGROUND: Microorganisms adhere to surfaces, creating biofilms with polysaccharides, fostering drug-resistant pathogens. These biofilms on surfaces and implants lead to persistent infections. Our study aims to detect biofilm formation and their antibiotic susceptibility pattern, shedding light on the development of chronic and challenging infections. MATERIAL METHODS: This cross-sectional study is carried out in the department of Microbiology Integral Institute of Medical Science & Research Lucknow. Totalsamples collected were 300 from various clinical samples and processed as per standard protocolsand identification of biofilm production was done using Congo Red Agar and Tube Method. RESULTS: Out of 300 positive samples 142 were gram positive and 158 were gram negative. In this study most frequently isolated organism wasE.coli followed by CONS, Staphylococcus aureus, Klebsiella sp., Enterococcus sp. and Pseudomonas sp. Biofilm detection by CRA method and Tube Method shows the result 47.7% and 50.3% respectively.