The yeast Lachancea thermotolerans is significant in winemaking due to its ability to produce L (D)-lactic acid, aiding in biological acidification and pH reduction. New strains were isolated from naturally fermenting fruits and assessed with regard to winemaking. Their physiological and fermentative characteristics were examined in various environments. Fermentation trials with synthetic must, synthetic base wine, and grape must showed that fermentation capacity, lactic acid production, and survival depended on the yeast strain and environmental conditions. Adding different concentrations of tartaric and sulphurous acids affected yeast viability and acidification ability. Treatment with 1 g/L tartaric acid and 50 mg/L metabisulfite resulted in the highest yeast imposition. Higher concentrations were toxic, causing yeast disappearance by the seventh day. Wines made with L. thermotolerans contained 1% less alcohol but had worse organoleptic characteristics and less aromatic complexity than the control wines. They also displayed an imbalance due to high acidity and low pH. The new strains show potential for winemaking, especially for biological acidification, but further research is needed to improve their organoleptic and aromatic qualities. Additionally, optimizing fermentation conditions and mixing with wines made with S. cerevisiae could enhance the overall quality and acceptance of wines produced with L. thermotolerans strains.