Lactic acid bacteria are frequently utilized in food industry and they are also recognized as antimicrobial agents due to their capability to produce metabolites such as: organic acids, biosurfactants, bacteriocins, hydrogen peroxide, cyclic dipeptides, exopolysaccharides. The main goal of this paper was to present the results of the research carried out on the strain LCM2 of lactic acid bacteria isolated from brined cucumbers, for production of biosurfactants and to assess its antifungal properties. The emulsification capacity of biosurfactant was measured using kerosene as the hydrophobic substrate. The value of emulsification index E24 was 89.04% showing a high emulsification activity of the biosurfactant. The structural characterization of biosurfactant by TLC revealed its glycolipidic nature. Assay of the ionic charge established the anionic charge of the biosurfactant revealed by the presence of precipitation lines towards the cationic surfactant dodecyl-dimethyl-ammonium chloride. The biosurfactant presented antibiofilm activity with low adherence capacity, structural damages of the hyphal net, conidiophores and delays or lack of sporulation and decreased biomass accumulation in four mycotoxigenic Penicillium and Aspergillus isolates. Results of in vitro assays recommend the biosurfactant produced by the new lactic acid bacteria strain LCM2 for biotechnological purposes, as alternative antifungal agent in food industry.