Two new canthin-6-one alkaloids, 4,9-dimethoxy-5-hydroxycanthin-6-one (1) and 9-methoxy-(R/S)-5-(1-hydroxyethyl)-canthin-6-one (2), together with fifteen known ones were isolated from the roots of ThailandEurycoma longifoliaJack. Among the known canthin-6-one alkaloids, compounds9and16were isolated from theEurycomagenus for the first time. Meanwhile, the nitric oxide (NO) inhibitory activities of all isolates were examined in lipopolysaccharide (LPS)-stimulated RAW264.7 cells at 50 mu M. Moreover, a dose-dependent experiment was conducted for active compounds1,2,4,6,7,10,12-17at the concentration of 10, 25, and 50 mu M, respectively. Consequently, compounds1,4,6,7,12,14,15, as well as17were found to inhibit NO release from RAW264.7 cells in a dose-dependent manner.