Sixty-five compounds were isolated from the roots of Eurycoma longifolia and characterized by comprehensive analyses of their 1D and 2D NMR, and mass spectral data. Among these isolates, four quassinoid diterpenoids were reported from natural sources for the first time, namely eurycomalide A (1), eurycomalide B (2), 13beta, 21-dihydroxyeurycomanol (3), and 5alpha, 14beta, 15beta-trihydroxyklaineanone (4). Screening of cytotoxicity, anti-HIV and antimalarial activity of these isolated compounds was also furnished by in vitro assays. Compounds 12, 13, 17, 18, 36, 38, 59, and 62 demonstrated strong cytotoxicity toward human lung cancer (A-549) cell lines, however, 12, 13, 17, 38, 57, 58, and 59 exhibited strong cytoxicity toward human breast cancer (MCF-7) cell lines. Compounds 57 and 58 displayed potent antimalarial activity against the resistant Plasmodium falciparum. The thorough studies on the stereochemistry of the different quassinoid diterpenoids provide a clear reference to the scientists who are interested on this field.