Ergosterol (ERG) is a bioactive sterol found in fungal cell membranes with reported cholesterol-lowering, antioxidant, and antitumor properties. Supercritical Fluid Extraction (SFE) conditions were optimized for Lentinula edodes (shiitake) using Response Surface Methodology and subsequently applied to Pleurotus ostreatus (oyster). Optimized SFE (690 bar, 69.8 °C, no co-solvent) produced significantly more concentrated ERG extracts than Soxhlet extraction for both species—280.57 ± 10.80 mg_ERG/g_extract for shiitake and 95.87 ± 7.18 mg_ERG/g_extract for oyster—corresponding to a 107% and 65% increase, respectively. Three biomass pre-treatments—ultrasound, microwave, and enzymatic—were evaluated in combination with SFE. Enzymatic pre-treatment with chitinase significantly improved ERG concentration: 337.53 ± 23.12 mg_ERG/g_extract for shiitake. These results obtained after analysis of samples by GC-MS demonstrate that high-pressure SFE combined with chitinase pre-treatment is an effective strategy for producing ERG-rich extracts from fungal matrices.