Background: Probiotic products contain metabolites that have positive effects on the intestinal microorganisms and well-being of the host as well as possess antimicrobial properties. Objective: This study aimed to investigate the antifungal activities of the water-soluble peptides (WSPs) found in ewe and cow milk KEFIR fermented by the traditional KEFIRan of Semnan (Semnan province, Iran). Materials and Methods: KEFIR samples were prepared by inoculating Iranian traditional KEFIRan into pasteurized milk. WPSs were extracted and antifungal activity was evaluated. Results: During the 28-day storage, the concentration of the amino acids increased except for a decrease detected in the concentration of arginine, aspartic acid, cysteine, and glycine,and the total amino acid concentration in ewe milk and KEFIR was higher than that in cow’, s milk and KEFIR. The WSPs of both KEFIR samples showed considerable inhibitory activities against the growth of Candida albicans and Aspergillus niger, but Penicillium sp. had the lowest sensitivity when treated with WSPs. The antifungal activity of WSPs of ewe KEFIR was significantly higher than that of cow KEFIR. The highest growth inhibitory potential of WSPs between two KEFIR samples was found for C. albicans. The antifungal potential of WSPs of ewe KEFIR was considerably higher than that of cow KEFIR. Conclusion: The higher antifungal potential of ewe KEFIR was likely associated with the high concentration of protein, extensive degradation of proteins, and diversity of amino acids produced during the fermentation.