Potentially probiotic yeasts isolated from foodstuffs can be used as components in functional fermented beverages. To date, there have been no reports on the use of Saccharomyces cerevisiae var. boulardii, Pichia kudriavzevii, Metschnikowia pulcherrima, or Hanseniaspora uvarum isolates in the production of a traditional Polish beverage called underbeer (podpiwek). The aim of the study was to determine the usefulness of six isolates of the above-mentioned species as starter cultures for the fermentation of underbeer. First, the important characteristics of the yeasts, like ethanol tolerance and H2S production, were examined. In the next stage, the wort was fermented by the tested yeasts, and cell viability, fermentation vigor, sugar assimilation, and production of metabolites, as well as properties of the beverage (pH, titratable acidity, color, and turbidity), were determined. Saccharomyces yeasts tolerated the addition of ethanol up to 16% (v/v), while Pichia, Metschnikowia, and Hanseniaspora tolerated up to 10% (v/v) ethanol, and all except H. uvarum produced H2S. The yeasts remained viable in the beverages for 1 month at the required level, utilized glucose, fructose and partially complex carbohydrates, and produced ethanol (S. cerevisiae, P. kudriavzevii, and M. pulcherrima) and organic acids such as tartaric, malic, and citric acid. The underbeers became sour and showed varying turbidity and a color corresponding to pale-amber beers. All tested strains produced fermented beverages that were low- or non-alcoholic with different properties. This experiment may be a starting point for research into regional products as probiotic or synbiotic foods; however, further research is required for selection of the best strains for underbeer fermentation.