The aim of this investigation was to evaluate the antifungal activity of Osmotin against several important economically improtant plant pathogenic fungi. The Osmotin gene from Nicotiana tabacum was overexpressed in Escherichia coli (Rosetta DE3). Taguchi test was applied to optimize the conditions for protein expression, and Western blot analysis confirmed expression of the recombinant protein. The expressed Osmotin was found in the form of insoluble inclusion bodies, which were then solubilized and refolded. The purified and refolded protein's activity was verified by three different antifungal activity assays. The purified recombinant Osmotin demonstrated a wide range of antifungal activity against various types of phytopathogenic fungi: Botrytis cinerea, Sclerotinia sclerotiorum, Fusarium oxysporum, Verticillium dahliae and Alternaria brassicola. Hemolysis biosafety test demonstrated that the protein is non-toxic to mammalian cells. All these findings suggest that the Osmotin gene from N. tabacum has promising potential as an antifungal agent against various phytopathogenic fungi and also to be utilized for production of antifungal agents and fungal-resistant transgenic plants.