Herein, a supercritical extraction plant (with a 6 L extraction cell) was successfully used to obtain ergosterol-enriched extracts from Lentinula edodes under the following conditions: a temperature of 40 °C, pressure of 225 bar, reaction time of 1-5 h, and the flow rate of 20 L h-1 for recirculated CO2. Moreover, ergosterol (ERG) and the SFE extract (SFE) with highest ergosterol concentration were microemulsified and submitted to in vitro digestion to study their ability to displace cholesterol from dietary mixed micelles (DMMs). ERG was also mixed with a β-glucan-enriched (33.5%) extract (BGE) obtained from L. edodes to investigate the synergies between them; the results indicated that all these extracts (including BGE without ERG) could reduce the cholesterol levels in the DMMs. However, when ERG and SFE were simultaneously administered to mice with a hypercholesterolemic diet, no significant differences in the serum cholesterol levels were detected as compared to the case of the control. However, when only BGE was administrated to another mice model previously induced with hypercholesterolemia, significant reduction in the cholesterol levels was noticed.