The gaseous reactive oxygen/nitrogen species (RONS) generated by cold atmospheric plasma (CAP) can effectively inactivateAspergillus flavus (A. flavus)and prolong the shelf-life of food. Plasma-activated water (PAW) is the extension of cold plasma sterilization technology. Without the limitation of a plasma device, PAW can be applied to more scenarios of food decontamination. However, the efficacy of PAW as a carrier of RONS for eradicatingA. flavusor inhibiting its growth remains unclear. In this study, the immediate fungicidal effect and long-term inhibitory effect of PAW onA. flavuswere investigated. The results demonstrated that 60-min instant-prepared PAW could achieve a 3.22 log reduction CFU/mL ofA. flavusand the fungicidal efficacy of PAW gradually declined with the extension of storage time. Peroxynitrite (ONOO−/ONOOH) played a crucial role in this inactivation process, which could damage the cell wall and membrane structure, disrupt intracellular redox homeostasis, and impair mitochondrial function, ultimately leading to fungal inactivation. In addition to the fungicidal effect, PAW also exhibited fungistatic properties and inhibited the synthesis of aflatoxin B1(AFB1) inA. flavus. By analyzing the cellular antioxidant capacity, energy metabolism, and key gene expression in the AFB1synthesis pathway, it was discovered that PAW can significantly reduce ATP levels, while increasing SOD and CAT activity during 5-d cultivation. Meanwhile, PAW effectively suppressed the expression of genes related to AFB1synthesis.