The contamination of peanuts, withAspergillus flavusand subsequent aflatoxins (AFs) is considered to be one of the most serious, safety problems in the world. Water activity (aw) and temperature are limiting, factors for fungal growth and aflatoxin production during storage. The objectives of this study were to integrate data on the effects of temperature (34, 37, and 42 °C) and water activity (aw; 0.85, 0.90, and 0.95) on growth rate aflatoxin B1 (AFB1) production and up- or-downregulation of the molecular expression of biosynthetic AFB1 genes divided into three types based on theirA. flavusisolate composition and AFB1 capacity in vitro:A. flavusKSU114 (high producer),A. flavusKSU114 (low producer), andA. flavusKSU121 (non-producer). TheA. flavusisolates were shown to be resilient in terms of growth on yeast extract sucrose agar media when exposed to temperature and water activity as pivotal environmental factors. The optimal conditions for the fungal growth of three isolates were a temperature of 34 °C and water activity of 0.95 aw; there was very slow fungal growth at the highest temperature of 42 °C, with different awvalues causing inhibited fungal growth. The AFB1 production for the three isolates followed the same pattern with one exception:A. flavusKSU114 failed to produce any AFB1 at 42 °C with different awvalues. All tested genes ofA. flavuswere significantly up- or downregulated under three levels of interaction between temperature and aw. The late structural genes of the pathway were significantly upregulated at 34 °C under aw0.95, althoughaflR,aflSand most of the early structural genes were upregulated. Compared to 34 °C with an awvalue of 0.95, most of the expressed genes were significantly downregulated at 37 and 42 °C with awvalues of 0.85 and 0.90. Additionally, two regulatory genes were downregulated under the same conditions. The expression level oflaeAwas also completely associated with AFB1 production, while the expression level ofbrlAwas linked toA. flavuscolonization. This information is required to forecast the actual effects of climate change onA. flavus. The findings can be applied to improve specific food technology processes and create prevention strategies to limit the concentrations of potential carcinogenic substances in peanuts and their derivatives.