Three extraction methods were sequentially combined to obtain fractions from Lentinula edodes (shiitake mushrooms) containing bioactive compounds against cardiovascular diseases (CVDs). Fruiting bodies were first extracted with plain water, obtained residue was then submitted to supercritical fluid extraction (SFE) and remaining residue submitted to hot water extraction. Sequential design allowed reutilization of the nonextracted material as raw material for the successive extractions increasing extraction yields and separating interesting compounds. Obtained fractions contained different amounts of ss-glucans, chitins, eritadenine, lenthionine, ergosterol, proteins/peptides and phenolic compounds conferring them different bioactivities. Water soluble fractions showed high antioxidant activities (ABTS(+center dot) and DPPH center dot scavenging capacity and reducing power), they were also able to inhibit one of the main enzymes involved in hypertension (angiotensin-I converting enzyme) and the key enzyme of cholesterol metabolism (3-hydroxy-3-methylglutaryl coenzyme A reductase). The latter inhibitory activity was also noticed in SFE extracts although ergosterol and other lipid-like molecules were isolated. Dietary fibers were separated in the third extraction. Therefore, with this sequential extraction procedure bioactive compounds against CVDs can be selectively separated from a single batch of shiitake powder. (c) 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:746-755, 2018