Eurycoma longifolia, native to Burma, Indochina, Thailand, and Southeast Asia is a tall Simaroubaceous slender shrub-tree commonly found as an understroy in the lowland forests at up to 500 m above sealevel. E. longifolia known locally as ‘Tongkat Ali’ in Malaysia, ‘Pasakbumi’ in Indonesia, ‘Cay ba binh’ in Vietnam and ‘Ian-don’ in Thailand is popularly sought after in herbal remedies and has been frequently prescribed either as a single ingredient or as a mixture with other herbs. The roots of this plant are used as folk medicine for the treatment of aches, persistent fever, tertian malaria, sexual insufficiency, dysentery, glandular swelling, and as health supplements. Recently, Lin et al. reported that the crude ethanolic extract of the root of E. longifolia could decrease the basal release of testosterone but increase the human chorionic gonadotropin (hcG)- induced production of testosterone by rat leydig cells. From the roots, several classes of compounds have been identified and they included quassinoids, canthin-6-one alkaloids, β-carboline alkaloids, tirucallane-type triterpenes, squalene derivatives, and biphenyl-neolignans. Some of these constituents were shown to possess cytotoxic, antimalarial, antiulcer, antipyretic, and plant growth inhibition activities. In addition, the crude extracts of this plant were reputed to increase male virility and sexual prowess and gained notoriety as a male aphrodisiac. The wide spectrum of pharmacological activities associated with the constituents and crude extracts of the title plant has further prompted us to undertaken the chemical investigation of the methanolic extract of the root of E. longifolia. In a previous paper, we reported the isolation and structural elucidation of cytotoxic canthin-6-one and b-carboline alkaloids. As a result of further fractionation efforts, two new C19-skeleton quassinoids, two new highly oxygenated klaineanone type C20 quassinoids, and sixty-one known compounds were isolated from the root of E. longifolia. Hence, we wish to report on the isolation and characterization of compounds (Fig. 1) and the cytotoxicity, antimalarial and anti-HIV activities.