Lignocellulosic biomass can be used as a source for energy, fuel and valuable chemical production. From all available technologies, biological approaches have been recognized as the most environmentally friendly and sustainable ones. At the same time, high conversion costs, low efficiency and environmental issues still hinder the introduction of biological processes into industrial scale manufacturing. The aim of this study was to determine the most suitable enzyme cocktail recovery conditions from a biomass–fungal culture of the white-rot basidiomycete Irpex lacteus. Subsequent evaluation of the overall enzyme cocktail efficiency to release fermentable carbohydrates from biomass showed that prolonged fungal cultivation decreases the quality of the produced enzyme cocktail. At the same time, introduction of ultrasound pre-treatment during enzyme extraction improved the recovered enzyme cocktail efficiency in converting biomass to fermentable sugars, yielding up to 0.25 g of fermentable sugar per g dry hay biomass and up to 0.11 g per g dried straw or microalgae substrates. The results demonstrated that the production of lignocellulose-degrading enzymes from fungi is more sensitive than previously described, especially in terms of fungal growth, culture sterility and incubation conditions.