Sesquiterpene lactones are a wide and various group of natural compounds showing a large spectrum of biological activities, including antitumor, anti-inflammatory, antibacterial and antifungal activities [1]. These compounds are mainly distributed in the Asteraceae family. The genus Ambrosia (Asteraceae) includes 41 species widespread in the temperate regions of the Northern Hemisphere and South America but many of which are adapted to the arid climates of the desert. The genus is best known for the severe and widespread allergies caused by its pollen. A. cumanensis Kunth is a medicinal plant native of Central and South America. The leaves are used in Colombian traditional medicine for stomach pain, intestinal infections, gastritis, and muscle aches. Previous biological studies on extracts obtained from A. cumanensis leaves, reported antimicrobial and spasmolitic activity [2]. On the basis of our previous studies on plants belonging to the Ambrosia genus [3, 4] and some biological activities reported, a phytochemical investigation on A. cumanensis aerial parts was performed. The dried powdered leaves, collected in Eastern Antioquia region of Colombia, were extracted with MeOH and partitioned between n-butanol/water to remove sugars. The n-BuOH extract was dried and dissolved in chloroform and subjected to different chromatographic techniques such as Silica gel, MPLC and RP-HPLC. Eleven sesquiterpene lactones, including three new natural products (1-3) were characterized by 1D- and 2D-NMR and MS analyses. New compounds were tested for their antiproliferative activity on HeLa (cervical cancer), Jurkat (T lymphocyte), and U937 (lymphoma) cell lines. Compound 3 (2, 3-dehydropsilostachyn C), was found to be the most active showing an IC50 6µM in Jurkat and 8µM in U937, respectively.