Background: In general, silver nanoparticles (AgNPs) are particles of silver with a size less than 100 nm. In recent years, synthesis of nanoparticles using plant extract has gained much interest in nanobiotechnology. In this concern, this study investigates green synthesis of AgNPs from silver nitrate using Sinapis arvensis as a novel bioresource of cost-effective nonhazardous reducing and stabilizing compounds. A stock solution of silver nitrate (0.1 M) was prepared. Different concentrations of silver nitrate (1, 2.5, 4, and 5 mM) were prepared from the above solution, then added to 5 mL of S. arvensis seed exudates. The mixtures were kept in 25°C. The synthesis of AgNPs was confirmed by the change in mixtures color from light yellow to brown. The antifungal activity of synthesized AgNPs was investigated in vitro. Results: The resulting AgNPs were characterized by UV-vis spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR). Formation of the AgNPs was confirmed by the change in mixture color from light yellow to brown and maximum absorption at 412 nm due to surface plasmon resonance of AgNPs. The role of different functional groups in the formation of AgNPs was shown by FTIR. X-ray diffraction was shown that the AgNPs formed in our experiments were in the form of nanocrystal, and TEM analysis showed spherical particles with an average size of 14 nm. Our measurements indicated that S. arvensis seed exudates can mediate facile and eco-friendly biosynthesis of colloidal-spherical AgNPs with a size range of 1 to 35 nm. The synthesized AgNPs showed significance antifungal activity against Neofusicoccum parvum cultures. Conclusion: The AgNPs were synthesized using a biological source. This synthesis method is nontoxic, eco-friendly, and a low-cost technology for the large-scale production. The AgNPs can be used as a new generation of antifungal agents.