Acetylenic acids, a class of fatty acid derivatives distinguished by acetylenic bonds, are widely distributed in medicinal plants and animals. These natural products exhibit a range of pharmacological properties, including insecticidal, antibacterial, antitumor, and antiviral effects. This article systematically reviews the active natural acetylenic acid compounds identified from natural resources between 1980 and 2024, with the aim of providing a consolidated reference to support future research and development in this field.