In search of a safe and new biosource for treating fungal infections, the bacterial lysate of probiotic Lactobacillus fermentum was tested for its antifungal activity against Aspergillus niger, Aspergillus flavus and Candida albicans. The antifungal activity of lysate was analyzed by dry weight, disc diffusion and micro broth dilution methods. The growth of A. niger and A. flavus was inhibited by 31 µg lysate protein/disc but C. albicans was firstly inhibited at 62 µg lysate protein/disc. MIC for inhibition of A. niger was recorded as 125 µg lysate/ml while, A. flavus and C. albicans were inhibited at MIC of 62 µg lysate/ml by micro broth dilution assay. De Man Rogose and Sharpe medium supplemented with wheat bran, corn steep liquer and yeast extract showed the highest yield, 10 g dry biomass/L of L.fermentum at pH 6.8. The elution profile of the purified lysate showed five fractions, the first gave more than 60% of the original sample. Its MIC was 25 µg lysate/disc. Its molecular weight was 20 to 30 KDa. Toxicity tests revealed that, up to 150 µg lysate/ml showed no significant haemolysis.