Polyacetylenes are a class of polyketides related to fatty acids that occur in plants, fungi, marine organisms and animals. These compounds show a pleiotropic profile of bioactivity, that includes antitumor, antibacterial, antimicrobial or antifungal properties. Because of this, the literature on these compounds has grown exponentially, and this review aims at summarizing the inventory of polyacetylenes occurring in terrestrial eukaryotic organisms (plants and fungi) during the last 15 years, and at discussing progress in their bioactivities and in the identification of their biological targets.