Abstract <p>Antifungal effect of chitosan aspartate shell nanoparticles, obtained by in situ self-assembly of protonated macrochains was investigated using 9 species of ascomycetes and 2 species of basidiomycetes of various physiological and ecological groups and artificial infection of soft wheat seedlings with a pathogenic fungus <i>Rhizoctonia</i> sp. in vitro. It was established that the biopreparation had antifungal activity against a wide range of soil-inhabiting saprotrophic and phytopathogenic fungi; the activity increased with an increase in the concentration of the dispersion of nanoparticles in the range of 0.001–0.1 g/dL. Inhibition of mycelial growth under the influence of nanoparticles was noted for fungi <i>Trichoderma harzianum</i> (up to 81.3%), <i>Fusarium oxysporum</i> (39.1%), <i>Schizophyllum commune</i> (37.9%), <i>Lecanicillum aphanocladii</i> (30.4%), <i>Alternaria</i> sp. (33.0%), <i>Botrytis</i> sp. (30.0%), <i>Trichoderma viride</i> (25.3%), <i>Sclerotinia</i> cf<i>. sclerotiorum</i> (18.0%), <i>Rhizoctonia</i> sp. (15.0%), <i>Talaromyces sayulitensis</i> (7.0%), and <i>Pleurotus ostreatus</i> var. Florida (6.1%). At the same time, a stimulating effect of low nanoparticle concentrations on the growth of ascomycete <i>T. sayulitensis</i> isolated from the rhizosphere (20%) was found. While <i>Rhizoctonia</i> sp. demonstrated in vitro higher resistance to the biopreparation compared to other phytopathogens, treatment of seeds with a dispersion of nanoparticles followed by cultivation on an artificial infectious background in the presence of fungal spores reduced the degree of damage and the level of plant disease development down to 33%. Possible mechanisms of the antifungal action of chitosan nanoparticles are discussed. The obtained data make it possible to propose the preparations based on chitosan aspartate nanoparticles dispersion as a safe biodegradable antifungal preparation in the practice of plant protecting from phytopathogenic fungi.</p>