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Biska, a traditional Istrian herbal spirit, is produced by macerating mistletoe leaves and twigs in a water–ethanol base, usually grape marc spirit. The aim of this study was to determine the optimal mistletoe maceration parameters (ethanol content, concentration of plant material, and duration) for the production of biska, which have not yet been the subject of scientific research. The prepared mistletoe macerates and the collected biska samples were subjected to phenolic compound characterization, including total phenolic content (TPC), total flavonoid content (TFC), and polyphenolic profile, by ultra-high-performance liquid chromatography coupled with mass spectrometry (UPLC-ESI-MS2). In addition, the macerates and the biska samples were analyzed for their biological properties: antioxidant and antiproliferative activity. The results showed that both the macerates and the biska samples contain phenolic compounds of mistletoe such as phenolic acids, flavonoids, and coumarins, which contribute to the observed biological activities—significant antioxidant activity and antiproliferative effect. By applying the maceration parameters, which yielded the highest content of phenolic compounds as well as volatile aromatic compounds (40–80 g/L of mistletoe, 40–55% v/v ethanol, and 21 days’ maceration time), the production process can be improved to produce biska with better aromatic and biological properties.

Maceration parameters such as alcohol and mistletoe content were studied to obtain high-quality macerates to produce biska, a traditional Istrian herbal spirit. An ethanol–water solution containing 25, 40, 55, and 70 v/v% ethanol and 20, 40, and 80 g/L mistletoe was macerated and pH, total acids, extract content, and color parameters were measured. Volatile compounds were determined by GC/MS. More than 150 different volatile compounds were detected. The composition of the ethanol–water solution and mistletoe content affects the values of total extract, total acids, and color. Samples with lower alcohol content have a higher number of different acids, aldehydes, alcohols, esters, and ketones, while macerates with higher alcohol content are rich in hydrocarbons and terpenes. Ethyl decanoate; ethyl hexynoate; ethyl octanoate; benzaldehyde; hexanal; octanal; and the terpene compounds limonene, cymene, menthone, humulene, eucalyptol, linalol, and borneol contribute significantly to the aroma of the macerates due to a low odor threshold. Ylangene and α- and β-bourbonene are tentatively determined new terpene molecules that are not found in the mistletoe literature, as well as thujone, which was also detected. For the preparation of aromatic macerates, 40–50 v/v% ethanol and about 40 g/L mistletoe should be used.