Many organic and inorganic compounds are known to exhibit widespread anti-fungal activity with successful usage in agricultural applications. The action mechanism of sulfur agents is that sulfur interacts with organic substances and forms sulfides and pentatonic acid having anti-microbial and anti-parasitic activities. Nowadays elemental sulfur is widely used in plant growing as a fungicide and acaricide. In recent years great considerable attention has been paid to the synthesis methods of antifungal properties of nanoscale sulfur due to the development of nanotechnology. Micronized sulfur and sulfur nanoparticles have antifungal and inbition effects of sporulation against pathogenic fungi. In this study, the antifungal activities in vitro of nano calcium polysulfide were investigated against two pathogenic fungi caused disease on tomato (Fusarium oxyporium lycopersici, Botrytis cinerea). Different concentrations of Nano calcium polysulfide (0,0.5, 1, 2%) were tested for growth inhibition of Fusarium oxyporium lycopersici, Botrytis cinerea. Growth inhibitions of tested pathogens were measured in vitro. Nano calcium polysulfide showed low inhibiton against Fusarium oxyporium lycopersici, Botrytis cinerea. Mycelial growth inhibition was observed at a maximum of 2% Nano calcium polysulfide application for Botrytis cinerea (34,4%) and 0.5% application for Fusarium oxyporium lycopersici (30.72). Nano calcium polysulfide inhibited only spore germination. Mycelial growth in both fungi was very weak increased in Nano calcium polysulfide compared to the control.