Macrofungi (mushrooms) have widely been appreciated all over the world for their nutritional values and medicinal properties. They have been used in traditional medicine for more than 3000 years for prevention and treatment of different diseases. Modern scientific research shows that macrofungi are producers of a broad spectrum of high- and low-molecular-weight bioactive compounds, i.e., alkaloids, polysaccharides, proteins, phenolics, terpenoids, polyketides, cyclic peptides, lectins and ribosome-inactivating proteins. They have various therapeutic effects as antidiabetic, anti-inflammatory, antimicrobial, antioxidant, antitumor, antiviral, cardioprotective, hepatoprotective, hypocholesterolemic, hypotensive, immunomodulatory, neuroprotective and regenerative, and possess promising pharmacological potential. Development of fungal biotechnological cultivation industry will support production of macrofungi-derived biotech products, healthy food and mycopharmaceuticals pharmaceuticals. Further advances in fungal biology and biotechnology, genomics and proteomics will assist biomedical research and application of macrofungi.