Natural compounds with bio-preservative properties are gaining attention of researchers worldwide. It is the case of biosurfactants known as surface active compounds produced by microorganisms which deserved antimicrobial and antioxidant properties. The present study was aimed to improve the shelf life of a Cameroonian indigenous fermented milk “pendidam” using biosurfactants derived from that food matrix. Biosurfactants was extracted from pendidam, purified, characterized preliminarly and its in vitro antimicrobial activity against microorganisms isolated from pendidam was assessed. Then, the biosurfactants was applied in pendidam and the microbiological quality of pendidam was followed up during 4 days of preservation at room temperature. The results obtained show that the crude extract of biosurfactants exhibit emulsifying and surface activities which remained stable after heat treatment at 100°C for 15 min, NaCl concentration of 20% and pH values ranging from 2 to 12. Furthermore, the biosurfactants was microbicidal against Candida spp. and E. coli at a concentration of 100 mg/mL. Solution of Biosurfactants (50 mg/mL) introduced into pendidam at a ratio of 10% (v/v), reduced significantly (p<0.05) the proliferation of the total aerobic mesophilic flora, E. coli and Candida spp. during 4 days of preservation at room temperature while no adverse effects were noticed on the growth of lactic acid bacteria. The present study highlights the bio-preservative potential of biosurfactants and suggests its use in food industry as food preservatives.