Yeasts are truly fascinating microorganisms. Due to their diverse and dynamic activities, they have been used to produce many interesting products, such as beer, wine, bread, biofuels and biopharmaceuticals. Saccharomyces cerevisiae (baker's yeast) is likely the most human-exploited yeast species. Saccharomyces is a popular choice for industrial applications, although its use in beer production dates to at least the sixth millennium BC. Baker's yeast represents a cornerstone of modern biotechnology, enabling the development of efficient production processes for antibiotics, biopharmaceuticals, technical enzymes and ethanol and biofuels. Today, diverse yeast species are explored for industrial applications, such as, e.g., Saccharomyces cerevisiae and Saccharomyces patorianus, hybrid brewer's yeast, Yarrowia lipolytica and Pichia fermentans. This installment-"Yeast Biotechnology 5.0"-continues the "Yeast Biotechnology" Special Issue series of the MDPI journal Fermentation. This Special Issue highlights prominent current research directions in the fields of yeasts as a cell factory (isocitric acid and cannabinoid production), yeast nanobiotechnology (yeast nanomotion), wine yeasts and wine fermentation, brewer's yeast and beer fermentation, yeasts for waste fermentation, coffee fermentation, distillery fermentation and ruminal fermentation and yeasts as probiotics.