Among the extremophilic yeasts, the osmotolerant, i.e., those that tolerate but have no absolute requirement for non-ionic solutes for their viability, and the osmophilic, i.e., those that have an absolute requirement for non-ionic solutes, are phylogenetically diverse, inhabiting both natural environments and foods with low water activity and high solute concentrations, such as sugar-rich foods—honey, dried fruits, and nectar. Mechanisms of osmotolerance include osmoregulation, maintenance of turgor pressure, and production of osmolytes, especially glycerol. Cultivation and isolation techniques for osmotolerant/osmophilic yeasts prevent osmotic shock by using specialized media and enrichment methods. In industry, these yeasts are important due to their role in food spoilage and food fermentations. Their applications also extend to biotechnology, where they contribute to the production of osmolytes and the enhancement of flavors in foods and beverages. Their unique properties also make them potential candidates for biotechnological advances.