Mushroom production is the biggest solid state fermentation industry in the world. Disposal and storage of spent mushroom compost (SMC) that remains after mushroom harvest poses a big economic and environmental problem. Production of industrially important hydrolytic enzymes by fungi on various agro-industrial wastes is a significant, open chapter in biotechnology. This paper proposes a novel application of SMC as substrate for cultivation of fungi in solid state fermentation (SSF) in order to obtain the enzymes cellulase, xylanase, amylase and β-glucosidase. SMC can be used as a good substrate for cultivation of Trichoderma and Aspergillus without the addition of supplementary (nutritive) elements. Starting amount of SMC was reduced by 30% due to hydrolysis by a complex of cellulolytic enzymes. Material that is left behind is a more suitable fertilizer for horticulture. One fungal isolate was pointed out as a promising producer (Trichoderma atroviride isolate T42). It produced the greatest amount of total protein (0.204 mg mL−1), five isoforms of β-glucosidase and the highest level (12 isoforms) of both endocellulase (0.76 U mL−1) and xylanase (2.31 U mL−1). The capacity of T42 to produce all examined enzymes in such a high number of isoforms demonstrates successful adaptation to new substrates.