This article focuses on anticancer, and other biological activities of acetylenic metabolites obtained from plants and fungi. Acetylenic compounds belong to a class of molecules containing triple bond(s). Naturally occurring acetylenics are of particular interest since many of them display important biological activities and possess antitumor, antibacterial, antimicrobial, antifungal, and immunosuppressive properties. There are of great interest for medicine, pharmacology, medicinal chemistry, and pharmaceutical industries. This review presents structures and describes cytotoxic activities of more than 100 acetylenic metabolites, including fatty alcohols, ketones, and acids, acetylenic cyclohexanoids, spiroketal enol ethers, and carotenoids isolated from fungi and plants.