The essential oil and methanol extract of Viola odorata L. were tested for antifungal activity against Botrytis cinerea in vitro on Petri plates and liquid culture, and under storage conditions. The essential oil composition of Viola odorata L. flowers were investigated by GC/MS with 63 identified volatile constituents. Phenyl butanone, linalool, benzyl alcohol, α-cadinol, globulol and viridiflorol were found to be the major components, respectively. The oil showed strong antifungal activity based on the inhibition zone and minimal inhibitory concentration values against the pathogen on Petri plates assays. The very low concentrations of them also reduced wet and dry mycelium weight of pathogen fungus in liquid culture. When the oils at 25, 12.5 and 6.25 μl/ml concentrations were applied to tomatoes before seven days of pathogen inoculation on storage conditions, the decay on fruits caused by the pathogen could be prevent completely. The results in this study showed that the essential oil of Viola odorata L. flowers had strong antifungal activity against pathogen fungi tested. So, the essential oil of Viola odorata L. could be used for management of this pathogen as a potential source of sustainable eco-friendly botanical fungicides.