The disaccharide trehalose plays a crucial role in multiple facets of the stress biology of yeasts and fungi. Here, we evaluate the properties, cellular and ecophysiological roles, metabolism, and stress-protection mechanisms of trehalose. We integrate disparate sources of knowledge across these topics, and bring new information about the mechanisms by which trehalose stabilises biomacromolecules and how trehalose metabolism is regulated thus giving rise to its diverse roles in fungi including stress protector, carbohydrate reserve, and regulatory/signaling molecule. We also present new findings about the effect of trehalose on microbial adaptation, complexity and spatio-temporal heterogeneity of microbial populations, and implications for industrial processes that apply fungi. Based on the elucidation of the structures of enzymes involved in trehalose synthesis, their catalytic mechanisms, and the regulation of trehalose synthesis, we discuss prospects for the development of more-efficient fungicides. Current humanitarian crises, such as overpopulation, global warming, malnutrition, immunocompromised conditions, and usage of immunosuppressant drugs, are making the incidence of human pathogens increases. Furthermore, fungal infections can be difficult to treat due to the conserved biochemistry between human and fungi cells. Serendipitously, however, trehalose is not synthesised by mammals, which makes trehalose synthesis an interesting target for the development of new therapies.