This study aimed to characterize the emulsifying activity of biosurfactants extracted from lactic acid bacteria (LAB) originals of the Argentinian Patagonia. Nine LABs were isolated from sheep cheese and fishes. All the strains produced cell-bound biosurfactants. Four selected strains produced biosurfactants able to decrease the air/water surface tension within the range of 28.1 mN/m–19.0 mN/m and exhibited an emulsification index after 24 h storage between 82 and 98%. An evaluation of the droplet size of emulsions revealed that the use of cell-free supernatant (CFS) obtained from Lactobacillus pentosus Tw226, Lactococcus lactis ssp lactis Tw12 and Lactococcus paraplantarum LbTw7 promoted the highest reduction in the Sauter and the De Broucker diameter demonstrating that the CFSs contained biosurfactants useful to form and stabilize oil in water emulsions. Moreover, a production of 0.26 ± 0.04 g of crude biosurfactant per gram of dry Lactobacillus pentosus Tw226 biomass was obtained. This yield was the highest registered among evaluated strains. Finally, thanks to its promising features it was selected for a preliminary characterization showing a critical micelle concentration of 0.11 g/L and a glycopolypeptide structure. This study demonstrated the potential uses of this biosurfactant as emulsifying agent in food products.