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The aim of this study was to determine the effects of environmental factors on the biofilm formation of Klebsiella pneumoniae , by allowing the organism to grow under different condition. The organism was grown in different media, pH, temperature, time, and substratum and in different body fluids. The amount of biofilm formed was determined using microtiter plate assay. Klebsiella showed more biofilm production in Tryptic soy broth than in Nutrient Borth, LB broth and BHI broth. Biofilm production was more at 40˚C than at 37˚C,42˚C and room temperature. Klebsiella showed comparable production of biofilms at pH 6 and 9 but maximum production was observed at pH 7.Biofilm formation was monitored for 36 hrs and maximum growth was observed between 18-20 hrs after that a gradual decrease was noted. Conditioning biofilm with urine showed maximum production than when blood and sputum were used. Similarly when substratum effects were checked rubber urinary catheter were found to be the best substratum when compared to polysterene ,PVC and hose . Thus we showed that biofilm formation by Klebsiella was influenced by host traits and environmental conditions at the site of colonisation and play an important role in medical device infections.