Bacteria are ubiquitous in nature, survive in all environment by forming biofilm on any suitable substrata. Biofilms can harbor human infectious agents in the environment as well in the human body. E.coli is a most important species that is found in the human gastrointestinal tract and has the ability to form biofilm both In vivo and In vitro. Biosurfactants play a significant role in biotechnology for industrial and medical applications. The surface hydrophobicity is modified by the adsorption of biosurfactants, thus interferes in the microbial binding and desorption processes. The production of biosurfactants by probiotic bacteria in vivo acts as a defense weapon against most troublesome pathogenic strains in the urogenital and gastrointestinal tracts and on medical devices. The biosurfactant shows promising results than the chemical surfactant as they are better biodegradable and do not pollute the environment. Lactobacillus is a probiotic bacterium which is recognized as potential interfering bacteria by producing various antimicrobial agents. In the current study, biosurfactant producing Lactobacillus sp. isolated from homemade curd sample. The TLC result indicates that it is a lipopeptide biosurfactant. The FTIR analysis predominantly shows the presence of alkene and alkyne groups. The antimicrobial activity of biosurfactant against E.coli is compared with different concentrations of chemical surfactant SDS and the results indicate a similar antimicrobial activity. The biofilm inhibition assays suggest that the biosurfactant produced by Lactobacillus inhibits the biofilm of E. coli.