Trichoderma harzianum T-33 and Trichoderma hamatum C-l are able to produce extracellular β-1,3-glucanases, chitinases, and cellulases in the shaked cultures, in the base medium enriched with mycelium (postcultural) or fodder yeast and supplemented with Avicel cellulose. The addition of fodder yeast (6%) and mycelium (6%) to the base medium induced more effective production of β-1,3-glucanases than the addition of lactose (2%). The production of chitinases and cellulases was the most successful in the medium enriched with mycelium. The enzymatic preparations from T. harzianum T-33 or T. hamatum C-l (obtained after ethanol precipitation) were used for Yarrowia lipolytica ATCC 20461 protoplast formation (57.5% or 74.5% after 4 h, respectively).