The role of volatile organic compounds (VOCs) in food, medicine, and agriculture is gaining significant attention. This comprehensive review delves into the biosynthetic pathways, regulatory and bioactivity profiles, and prospects of VOCs, encompassing a spectrum from volatile terpenoids to phenolics, aldehydes, ketones, and acids. We commence with an exploration of the diverse biosynthetic routes of VOCs, focusing on the methylerythritol phosphate, mevalonate, and phenylpropanoid pathways and elucidating the key enzymes and intermediates. Subsequently, we examine the bioactivity of VOCs, highlighting their antibacterial, anticancer, anti-inflammatory, antioxidative, anthelmintic, and anti-mood disorder properties, and discuss their potential applications in the food, medicinal, and agricultural fields. This review highlights the need for future studies to focus on regulating the biosynthesis of VOCs, unraveling their bioactivity, and developing efficient synthetic and extraction methods to enable sustainable advancements in related fields.