In a continuing search for novel anticancer principles from higher plants, the Tanzanian plant Acanthospermum glabratum (DC.) Wild (a member of the subtribe Melampodiinae, tribe Heliantheae in the family Asteraceae) exhibited anticancer activity both in vitro and in vivo. Separation of the constituents with coincidental in vitro bioassay of all appropriate fractions led to the isolation of nine constituents displaying cytotoxic activity. The plant yielded nine cytotoxic compounds, seven of which are new sesquiterpene lactones in the melampolide series. Structures of the new compounds were assigned based on spectra comparison with acantholide (3), a compound whose structure was determined by X-ray analysis. New compounds isolated in addition to acantholide (3) were acanthamolide (12), acanthospermolide (1), glabratolide (5), 9-hydroxyglabratolide (6), acanthoglabrolide (7), and dihydroacanthospermal A (10), and the known compounds were acanthospermal A (8) and 3,6-dimethoxy-4',5,7-trihydroxyflavone (15).