Economic loss of postharvest wheat under poor storage conditions due to fungal spoilage and mycotoxin contamination is severe. In order to study the influencing factors of the aggravation of mildew in natural wheat during storage, we assessed changes in mycotoxins by high performance liquid chromatography, changes in fungal communities by high-throughput sequencing, and changes in biochemical components in wheat stored under artificial simulation conditions. Deoxynivalenol was the dominant mycotoxin, reaching 1103 μg/kg at 25 °C with 75% relative humidity after 30 weeks. Under these conditions, dominated the fungal communities, and was significantly negatively correlated with glutenin ( < 0.05). Low storage temperatures and low humidity result in lower levels of mycotoxins. Different fungi tended to consume different wheat components, and the interaction between environmental and biological factors eventually leads to the deterioration of wheat quality. These findings might provide valuable information for control strategies of mildew occurrence during grain storage.