The rapid development of biogas plants in China has generated large quantities of digestate. The disparity between the continuity of biogas plant operation and the seasonality of digestate utilization has led to the need to store digestate. Therefore, untargeted profiling of bioactive compounds in the digestate stored under aerobic and anaerobic conditions was performed. The antioxidant and antifungal activity of digestate stored under varying conditions was likewise assessed. The results delineated that digestate storage under varying conditions brought about the degradation of organic acids, alkenes, aldehydes, alcohols, ketones, ethers, amino acids and their derivatives, and esters, leading to the stabilization of digestate components. Together, these new data revealed that digestate storage for up to 20 days under aerobic conditions promotes glycine, serine, and threonine degradation pathways and enhances biotin and vitamins production. In contrast, anaerobic storage enhances the taurine and hypotaurine metabolic pathways and increases the derivation of antimicrobial substances, such as indole alkaloids. Moreover, digestate storage under anaerobic conditions promotes antioxidant and antifungal activity more than storage under aerobic conditions. These findings can contribute to the future development of high-value agricultural products from digestate and the sustainability of biogas plants. Further studies are required for the untargeted metabolomic of digestate under storage to explore the underlying mechanisms of promoting disease resistance by the digestate upon land application. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.