The antifungal activity of some lactic acid bacteria and Propionibacterium thoenii against some yeasts and moulds was tested by using well agar diffusion assay. Also, the nutritional requirements for the synthesis of antifungal substances (AFS) by tested culture were investigated. Out of the tested cultures, the most promising cultures having a broad spectrum of antifungal activity was Lactobacillus reuteri followed by Lactobacillus acidophilus. A combination between pairs of AFS was found to be the most active against tested yeasts and moulds. Among six media tested, trypticase soy broth was found to be the best medium for production of antifungal substances by L. reuteri and L. acidophilus. While, Elliker broth and sodium lactate broth supported maximum production of AFS by Lactobacillus rhamonsus, Pediococcus acidilactici and Prop. thoenii. Optimum antifungal activity was noticed when L. reuterii grown at 37°C for 16 h. Organic nutrients such as glucose and yeast extract, as well as NaCl and CaCl₂ influnced the production of antifungal substances when added to the growth medium. Each stimulated production at some concentrations. The efficacy of antifungal substances was determined to control the yeast and mould in Domiati cheese over long storage (21 d) at 7°C. Shelf-life analysis demonstrated that incorporation of antifungal substance produced by L. reuteri at a level of 10% could effectively inhibit the growth of Saccharomyces cerevisiae and Penicillum spp.