This study investigates the effect of maceration and different winemaking techniques on the species diversity of yeasts in white wines from the Slovak Tokay wine region, known for its traditional white wine production. Lipovina grape variety samples were divided into three groups: control (C), macerated (M) and macerated with the addition of a yeast culture (MY). During the entire fermentation process, quantitative and qualitative microbiological analyses of the raw material and must samples were carried out, which resulted in the identification of 60 yeast isolates via the API 20 C AUX biochemical test and MALDI-TOF MS. Identification was further verified via Sanger sequencing of PCR amplicons, which confirmed the presence of less common wild yeasts in Tokay wine must samples, including Aureobasidium pullulans, Cryptococcus magnus, Torulaspora delbrueckii and Rhodotorula sp. The highest species diversity was observed in the macerated group. These findings indicate that the quality and distinctiveness of Slovak Tokay wines can be increased by careful management of the maceration process during winemaking procedures.