Food-borne Fungi, both yeasts and moulds, play an undeniable role in the deterioration of the quality and hygine of foodstuffs due to their mycellial growth and/or production of mycotoxins. Development of fungal resistance to chemical preservatives besides consumer demand for minimally processed foods and reduced use of chemical preservatives, have stimulated researchs on biopreservatives. Biopreservation, refers to extended shelf-life and enhanced safety of foods, obtained by using the microorganisms and their metabolites. Among natural biological antagonists, Lactic Acid Bacteria (LAB), with GRAS status and the ability to produce variety of antimicrobial compounds, are of much importance. In this project, 42 isolates of Lactobacillus delbrueckii su. bulgaricus and 20 isolates of Streptococcus thermophilus, previously isolated from traditional yoghurts of Isfahan region, were screened for antifungal activity against Penicillium italicum , as the indicator mould, using Agar Overlay assay. The isolate “A” of Lactobacillus bulgaricus , as the most powerful antifungal isolate of all, was subjected to examinations for the evaluation of fungal inhibition spectra and production kinetics of antifungal metabolites. Effect of the type of culture medium and sodium acetate content on antifulngal activity and chemical nature of antifungal metabolites were also determined. Thirty nine out of 42 Lactobacillus bulgaricus isolates presented antifungal activity by developing inhibition halos while non of Streptoccous thermophilus isolates could create such halos. Results showed that the isolate “A” of Lactobacillus bulgaricus has a broad spectrum inhibitory activity against a variety of fungi species including Aspergillus and Penicillium species. The production of antifungal metabolites by the isolate, was shown to start at late logarithmic phase and to accelerate during stationary and death phases of growth. MRS was the best growth media for the isolate in order to exhibit its antifungal activity, which was also directly affected by sodium acetate content of the culture medium. Elimination of sodium acetat from MRS, led to complete loss of antifungal activity whereas high levels of the compound in both MRS and Tomato Juice Agar, resulted in amplified antifungal activity of the isolate. Antifungal effect of the isolate, was shown to be only partially affected by severe heat tratment as the cell-free supernatant of the isolate, retained more than 65% of its original antifungal activity after being heated for 1 second at 135°C (UHT sterilization) . Organic acids comprising Lactic, acetic and propionic acid, were synthesized by the isolate to the respective level of 202, 78 and 13 mM/L as Propionic acid showed the highest fungal inhibitory effect. These three organic acids, along with peptide compounds, were determined as the most significant antifungal metabolites, produced by the isolate. Keywords: Antifungal activity, Screening, Lactobacillus bulgaricus , Organic Acids, Peptide Compounds