Lactobacillus species produce biosurfactants that can contribute to the bacteria's ability to prevent microbial infections associated with urogenital and gastrointestinal tracts and the skin. Here, we described the biological and physicochemical properties of biosurfactants produced by Lactobacillus jensenii P.sub.6A and Lactobacillus gasseri P.sub.65. The biosurfactants produced by L. jensenii P.sub.6A and L. gasseri P.sub.65 reduced the water surface tension from 72 to 43.2 mN m.sup.-1 and 42.5 mN m.sup.-1 as their concentration increased up to the critical micelle concentration (CMC) values of 7.1 and 8.58 mg mL.sup.-1, respectively. Maximum emulsifying activity was obtained at concentrations of 1 and 5 mg mL.sup.-1 for the P.sub.6A and P.sub.65 strains, respectively. The Fourier transform infrared spectroscopy data revealed that the biomolecules consist of a mixture of carbohydrates, lipids and proteins. The gas chromatography-mass spectrum analysis of L. jensenii P.sub.6A biosurfactant showed a major peak for 14-methypentadecanoic acid, which was the main fatty acid present in the biomolecule; conversely, eicosanoic acid dominated the biosurfactant produced by L. gasseri P.sub.65. Although both biosurfactants contain different percentages of the sugars galactose, glucose and ribose; rhamnose was only detected in the biomolecule produced by L. jensenii P.sub.6A. Emulsifying activities were stable after a 60-min incubation at 100 degreesC, at pH 2-10, and after the addition of potassium chloride and sodium bicarbonate, but not in the presence of sodium chloride. The biomolecules showed antimicrobial activity against clinical isolates of Escherichia coli and Candida albicans, with MIC values of 16 microg mL.sup.-1, and against Staphylococcus saprophyticus, Enterobacter aerogenes and Klebsiella pneumoniae at 128 microg mL.sup.-1. The biosurfactants also disrupted preformed biofilms of microorganisms at varying concentrations, being more efficient against E. aerogenes (64%) (P.sub.6A biosurfactant), and E. coli (46.4%) and S. saprophyticus (39%) (P.sub.65 biosurfactant). Both strains of lactobacilli could also co-aggregate pathogens. This report presents the first characterization of biosurfactants produced by L. jensenii P.sub.6A and L. gasseri P.sub.65. The antimicrobial properties and stability of these biomolecules indicate their potential use as alternative antimicrobial agents in the medical field for applications against pathogens that are responsible for infections in the gastrointestinal and urogenital tracts and the skin.