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.