Spontaneously fermented wines are a habitat for many Saccharomyces and non-Saccharomyces strains that are typical for a given region. The isolates obtained can serve as regional starter cultures for winemaking. The aim of this study was to isolate, identify and evaluate the oenological properties and fermentation suitability of selected yeast isolates obtained from Polish spontaneously fermented grape wines. The isolated yeasts were genetically identified and characterised in terms of ethanol tolerance, enzymatic activities, H2S production, and preliminary killer activity. In small-scale fermentations conducted in CDGJ medium and grape juice, the fermentation rate, pH, number of yeast, content of sugars, ethanol, organic acids and volatile compounds were determined. Genetic identification revealed the species: Saccharomyces cerevisiae, S. paradoxus, Metschnikowia pulcherrima, M. ziziphicola, Hanseniaspora uvarum, and Pichia kluyveri. Non-Saccharomyces and Saccharomyces strains grew poorly in the presence of 4–6% (v/v) and 14–16% (v/v) ethanol, respectively. The yeasts had varied enzymatic activities in API ZYM tests, and production of H2S, but did not exhibit killer activity. The monocultures showed differences in fermentation rates. The best growth was recorded for all strains during grape juice fermentation, up to 109 cfu/mL, producing ethanol and glycerol in the range of 53.92–86.54 g/L and 0.0–4.48 g/L. Yeasts produced characteristic volatile compounds, e.g., esters: 2-phenylethyl acetate and ethyl decanoate. The monocultures of isolated yeasts can be used in fermentation of grape must, yielding wines with diverse characteristics in terms of ethanol, organic acids and volatile compounds.