The aim of this work was to prepare a softwood substrate on which to grow edible and medicinal mushrooms. Liquid digestate from a biogas station was successfully used in spruce sawdust fermentation. Pleurotus ostreatus, P. eryngii, and Ganoderma lucidum were grown on the obtained substrates and their mycelia grew at rates similar to rates of growth on control beech sawdust; values ranged from 4.1 to 5.54 mm/day. A 6-week fermentation period was determined to be sufficient for removing volatile extractives from sawdust (76% removal efficiency), which elevated content was shown to be most critical for fungal growth. Removal of 47% of resinous compounds and a decrease in the carbon-to-nitrogen ratio in the growth substrate were found during sawdust fermentation in the presence of the liquid digestate. Among ligninolytic enzymes, the growth substrates produced here favored laccase produced by tested fungi. It follows that utilizing wastes from biogas production to reuse softwood wastes could make an environmentally friendly and economically viable biotechnology for producing mushrooms.