Finding novel ways to avoid the economic impacts in agriculture brought on by phytopathogenic fungus has become necessary as a result of the constant requirement for supplying global food demand. Furthermore, the increased virulence of microbes against antimicrobial drugs has made the development of innovative compounds with broader action and less toxicity necessary. In this study, selenium nanoparticles (Se NPs) were biosynthesized by simple, cost-effective, environmentally method using Fusarium fujikuroi MED14. The optimum conditions for Se NPs biosynthesis were recorded at mixing of 4 discs with diameter 5mm from the biomass of fungal strain (grown on malt extract glucose yeast extract peptone) with 0.16 M of Na2SeO3 solution at 40℃ and dark conditions. The biosynthesized Se NPs were detected at a unique absorption peak ≈246nm. The optimized Se NPs appeared to be crystalline and uniformly distributed NPs varying from 52 nm to 68 nm in diameter with surface charge of +32.3 mV. The Se NPs showed antifungal activity against F. solani, Aspergillus niger, F. fujikuroi MED14 and Candida albicans with inhibition zones 20, 15, 13, and 15 mm, respectively. Therefore, this work provides an initial basis for further research employing F. fujikuroi MED14 in the green synthesis of antifungal Se NPs under various trial conditions.