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The present study was aimed to examine the use of culture filtrates of defensin-producing fungal strains against the most important fungal and pseudofungal infections of grapevine as an environmentally sound alternative of chemical control. Twenty-seven isolates of five defensin-producing fungal species were tested for antifungal activity. Four isolates (one Aspergillus giganteus and three Aspergillus niger) decreased the viability of the test organism successfully. The isolate with the highest antifungal efficacy (Aspergillus niger SZMC2759) was subjected to detailed investigation. The culture filtrate of the strain was effective against epicuticular mycelia and conidia of Botrytis cinerea, Guignardia bidwellii and Erysiphe necator, but not against subcuticular mycelia and Plasmopara viticola cells. The culture filtrate also prevented the development of powdery mildew symptoms on grapevine cuttings. Several modes of action were detected: inhibition of germination, membrane permeabilization, plasmolysis, fragmentation of nuclei. Certain parameters (pH, stress, aeration) of the fermentation were optimized for the highest antifungal activity. According to the results, culture filtrate of Aspergillus niger can be a promising agent in the management of fungal diseases of grapevine.