In this work, the extraction, structural analysis, and identification as well as antimicrobial, anti‐adhesive, and antibiofilm activities of lipopeptides produced by Enterobacter cloacae C3 strain were studied. A combination of chromatographic and spectroscopic techniques offers opportunities for a better characterization of the biosurfactant structure. Thin layer chromatography (TLC) and HPLC for amino acid composition determination are used. Efficient spectroscopic techniques have been utilized for investigations on the biochemical structure of biosurfactants, such as Fourier transform infrared (FT‐IR) spectroscopy and mass spectrometry analysis. This is the first work describing the production of different isoforms belonging to kurstakin and surfactin families by E cloacae strain. Three kurstakin homologues differing by the fatty acid chain length from C10 to C12 were detected. The spectrum of lipopeptides belonging to surfactin family contains various isoforms differing by the fatty acid chain length as well as the amino acids at positions four and seven. Lipopeptide C3 extract exhibited important antibacterial activity against Gram‐positive and Gram‐negative bacteria, antifungal activity, and interesting anti‐adhesive and disruptive properties against biofilm formation by human pathogenic bacterial strains: Salmonella typhimurium, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus cereus, and Candida albicans.