Yeasts are truly fascinating microorganisms. Due to their diverse and dynamic activities, they have been used for the production of many interesting products, such as beer, wine, bread, biofuels, and biopharmaceuticals. Saccharomyces cerevisiae (brewers’ or bakers’ yeast) is the yeast species that is surely the most exploited by humans. Saccharomyces is a top-choice organism for industrial applications, although its use for producing beer dates back to at least the 6th millennium BC. Bakers’ yeast has been a cornerstone of modern biotechnology, enabling the development of efficient production processes. Today, diverse yeast species are explored for industrial applications. This Special Issue “Yeast Biotechnology 2.0” is a continuation of the first Special Issue, “Yeast Biotechnology” (https://www.mdpi.com/books/pdfview/book/324). It compiles the current state-of-the-art of research and technology in the area of “yeast biotechnology” and highlights prominent current research directions in the fields of yeast synthetic biology and strain engineering, new developments in efficient biomolecule production, fermented beverages (beer, wine, and honey fermentation), and yeast nanobiotechnology.