To detect mildew in lotus seeds during storage and processing, gas chromatography-ion mobility spectrometry (GC-IMS) was applied to analyze the volatile organic compounds (VOCs) in the fungal infection process of lotus seeds through accelerated in vitro mildew infection simulation. Seven strains of fungi were isolated and identified from naturally contaminated lotus seeds. Next, accelerated in vitro mildew infection of normal lotus seeds was carried out by inoculation with Aspergillus tamarii, Aspergillus pseudoglaucus, Penicillium citrinum, and Penicillium cinnamopurpureum. The GC-IMS results reveal a total of 73 VOCs, mainly esters, alcohols, aldehydes, ketones, and acids. The study employed cluster heat map and principal component analysis to explore the differences and similarities among the various VOCs. The VOCs were found to vary widely across the different stages of fungal infection. Ethyl lactate, 1,3-butanediol, and isoamyl butyrate were proven to be potential biomarkers for early mildew detection in lotus seeds. This study aimed to evaluate the role of VOCs in the monitoring and early warning of lotus seed mildew. These findings have important implications for the storage and quality control of lotus seeds.