Aflatoxins produced by some species of Aspergillus are considered secondary toxic fungal by-products in feeds and food. Over the past few decades, many experts have focused on preventing the production of aflatoxins by Aspergillus ochraceus and also reducing its toxicity. Applications of various nanomaterials in preventing the production of these toxic aflatoxins have received a lot of attention recently. The purpose of this study was to ascertain the protective impact of Juglans-regia-mediated silver nanoparticles (AgNPs) against Aspergillus-ochraceus-induced toxicity by exhibiting strong antifungal activity in in vitro (wheat seeds) and in vivo (Albino rats) settings. For the synthesis of AgNPs, the leaf extract of J. regia enriched with high phenolic (72.68 ± 2.13 mg GAE/g DW) and flavonoid (18.89 ± 0.31 mg QE/g DW) contents was used. Synthesized AgNPs were characterized by various techniques, including TEM, EDX, FT-IR, and XRD, which revealed that the particles were spherical in shape with no agglomeration and fine particle size in the range of 16–20 nm. In vitro antifungal activity of AgNPs was tested on wheat grains by inhibiting the production of toxic aflatoxins by A. ochraceus. According to the results obtained from High-Performance Liquid Chromatography (HPLC) and Thin-Layer Chromatography (TLC) analyses, there was a correlation between the concentration of AgNPs and a decrease in the production of aflatoxin G1, B1, and G2. For in vivo antifungal activity, Albino rats were administrated with different doses of AgNPs in five groups. The results indicated that the feed concentration of 50 µg/kg feed of AgNPs was more effective in improving the disturbed levels of different functional parameters of the liver (alanine transaminase (ALT): 54.0 ± 3.79 U/L and aspartate transaminase (AST): 206 ± 8.69 U/L) and kidney (creatinine 0.49 ± 0.020 U/L and BUN 35.7 ± 1.45 U/L), as well as the lipid profile (LDL 22.3 ± 1.45 U/L and HDL 26.3 ± 2.33 U/L). Furthermore, the histopathological analysis of various organs also revealed that the production of aflatoxins was successfully inhibited by AgNPs. It was concluded that the harmful effects of aflatoxins produced by A. ochraceus can be successfully neutralized by using J. regia-mediated AgNPs.