The 50 % aqueous ethanol extract of Eurycoma longifolia Jack (Simaroubaceace) roots was partitioned with diethyl ether, n-butanol and then water. Acute toxicity studies of each fraction on mice administered orally and brine shrimps revealed that the n-butanol fraction was the most toxic. Toxicity-guided chromatographic fractionation of the n-butanol fraction identified eurycomanone as the most toxic component. 13,21-Dihydroeurycoma-none, eurycomanol, longilactone, 14,15β-dihydroxyklaineanone and eurycomanol-2-O-β-glucopyranoside were 2.8, 33, 44, 88.9 and > 100 times less toxic on brine shrimps, respectively. A C20-type quassinoid, an α,β-unsaturated ketone in ring A, an exomethylene function at C-13 and an oxymethylene bridge connecting C-8 and C-11 of ring C contributed to increased toxicity.