The co-occurrence of AFB1, AFB2, AFG1, and AFG2 in mold-contaminated feed suggests a significant contribution of A. parasiticus to aflatoxin contamination in feed. In contrast to A. flavus, research on the effects of A. parasiticus growth and aflatoxin production is limited. This study aims to investigate the factors influencing the growth and aflatoxin production by A. parasiticus, as well as to analyze the interactions among these factors utilizing response surface methodology. The results will be compared with relevant studies on A. flavus, ultimately contributing to the development of a comprehensive strategy for the prevention of aflatoxin contamination. The findings indicated that both A. parasiticus and A. flavus used sugar as an initial substrate to produce aflatoxins. The substrate preference of A. parasiticus was not changed due to differences in nutrient content in different feeds. The mineral Mn was found to suppress aflatoxin production by both fungi, whereas glucose, sucrose, and Zn stimulated production. Temperature and water activity levels effective against A. flavus may not be equally effective for A. parasiticus. The optimum storage conditions were for temperature and water activity below 10 °C and 0.85 at the same time. Although chemical pesticides may comply with regulatory limits in feed, certain pesticides and their interactions could increase the risk of aflatoxin contamination during feed storage. Hence, a comprehensive evaluation of factors influencing both fungi is imperative to prevent aflatoxin contamination effectively.