Edible and medicinal mushrooms contain bioactive compounds with promising effects on several cardiovascular risk biomarkers. However, strains of Ganoderma lucidum of Mexican origin have not yet been studied. Standardized extracts of G. lucidum (Gl) were given to C57BL/6 mice fed a high-cholesterol diet compared with the drug simvastatin. The effects of the extracts on serum biochemical parameters, liver lipid content, cholesterol metabolism, and the composition of gut microbiota were assessed. Acetylsalicylic acid (10 mM) added to the cultivation substrate modulated properties of Gl extracts obtained from mature basidiomata. Compared to the high-cholesterol diet group, the consumption of Gl extracts significantly reduced total serum cholesterol (by 19.2% to 27.1%), LDL-C (by 4.5% to 35.1%), triglyceride concentration (by 16.3% to 46.6%), hepatic cholesterol (by 28.7% to 52%) and hepatic triglycerides (by 43.8% to 56.6%). These effects were associated with a significant reduction in the expression of lipogenic genes (Hmgcr, Srebp1c, Fasn, and Acaca) and genes involved in reverse cholesterol transport (Abcg5 and Abcg8), as well as an increase in Ldlr gene expression in the liver. No significant changes were observed in the gene expression of Srebp2, Abca1 or Cyp7a1. In several cases, Gl-1 or Gl-2 extracts showed better effects on lipid metabolism than the drug simvastatin. A proposed mechanism of action for the reduction in cholesterol levels is mediated by α-glucans and β-glucans from Gl, which promoted decreased absorption of cholesterol in the gut, as well as greater excretion of fecal bile acids and cholesterol. The prebiotic effects of Gl-1 and Gl-2 extracts modulated the composition of gut microbiota and produced an increase in the Lactobacillaceae family and Lactobacillus genus level compared to the control group, high-cholesterol diet group and group supplemented with simvastatin. Mexican genetic resources of Gl represent a new source of bioactive compounds showing hypocholesterolemic properties and prebiotic effects.