Forty standard Lactobacillus cultures were checked for their antifungal properties against nine standard fungi. Yeasts were found to be more resistant than moulds. Lactobacillus brevis NCDC 02 was selected for further studies due to the maximum antifungal activity and proteinaceous nature of antifungal substance production. The optimum production of antifungal substances was at 37°C for 48 h and at the initial pH of 6–7 of the medium. The antifungal substances were heat- and pH-stable. The antifungal substances were found to be hydrophobic and their molecular weight was found to be more than 1 kDa and less than 5 kDa.