The MeOH-soluble fraction of the water extract of Catharanthus roseus from Indonesia, having shown potent inhibitory activity on the metabolism mediated by CYP2D6, was subjected to activity-guided isolation to yield two triterpenes, ursolic acid (1) and oleanolic acid (2), and three alkaloids, vindoline (3), ajmalicine (4), and serpentine (5). The isolated compounds were tested for their inhibitory activity on the metabolism mediated by CYP3A4 or CYP2D6 using [N-methyl-¹⁴C]erythromycin or [O-methyl-¹⁴C]dextromethorphan as a substrate, respectively. Ajmalicine (4) and serpentine (5) showed very potent inhibitory activity against CYP2D6 with IC₅₀ values of 0.0023 and 3.51 μM, respectively. All isolated compounds showed weak or no inhibition against CYP3A4. On time-, concentration-, and NADPH-dependent assay, serpentine (5) appear to be the mechanism-based inhibitor for CYP2D6 enzyme in which the inhibition was irreversible and driven by catalytic process. K_I and k_inact values for serpentine (5) were 0.148 μM and 0.090 min⁻¹, respectively. On the other hand, ajmalicine (4) showed no time-dependent inhibition or reversible inhibition, and thus appear to be not mechanism-based inhibitor.