Peanuts and corn are economically important crops that are frequently infected by Aspergillus flavus, Sclerotium rolfsii, and Fusarium species. In this study, atoxigenic A. flavus PA51 and PA61 were isolated, and both effectively inhibited aflatoxin B1 (AFB1) production in corn (up to 92.13 % and 89.21 %) and peanuts (up to 85.69 % and 95.77 %). At a concentration of 1 × 107 spores/mL, PA51 exhibited inhibition rates of 74.57 %, 88.67 %, and 90.44 % against S. rolfsii, Fusarium proliferatum, and Fusarium verticillioides, respectively, while PA61 achieved inhibition rates of 78.52 %, 86.59 %, and 90.59 %. Additionally, 20 % PA51 supernatant exhibited inhibition rates of 41.58 %, 50.84 %, and 43.25 % against S. rolfsii, F. proliferatum, and F. verticillioides, while PA61 achieved inhibition rates of 45.75 %, 40.90 %, and 45.55 %. For S. rolfsii, active compounds such as kojic acid in the supernatant inhibited sclerotia germination, oxalic acid secretion, and polygalacturonase activity. For Fusarium species, the supernatant significantly reduced fumonisin B1 (FB1) production, with PA51 (44.08 %-78.06 %) exhibiting stronger inhibition compared to PA61 (41.96-71.40 %). Transcriptomic and indicator analyses revealed that atoxigenic A. flavus exerts antifungal effects by disrupting cell wall and membrane integrity, inducing oxidative stress, and impairing energy metabolism. Field experiments demonstrated that the application of atoxigenic A. flavus reduced AFB1 and FB1 levels in peanuts, corn, and field soil by nearly 80 %. Additionally, it significantly reduced the abundance of pathogenic fungi, including Fusarium species, Ascomycota, and Basidiomycota. These findings indicated that atoxigenic A. flavus has significant potential for biological control and provided new insights into its antifungal mechanisms.