AbstractEurycoma longifolia(EL) andEurycoma harmandiana(EH) are natural medicinal plants belonging to the Simaroubaceae family, and are well‐known for their ability to enhance male sexual performance. The present study investigated the phosphodiesterase‐5 (PDE‐5) inhibitory activity of intact roots of EL and EH. Additionally, canthin‐6‐one alkaloids,β‐carboline alkaloids, and quassinoids were also screened for PDE‐5 inhibitory activity. We developedin vitroroot and callus cultures of EL and EH to determine their PDE‐5 inhibitory activity. Our results indicated that canthin‐6‐one alkaloids, which include canthin‐6‐one‐9‐O‐β‐D‐glucopyranoside, 9‐methoxycanthin‐6‐one, canthin‐6‐one, and 9‐hydroxycanthin‐6‐one, exhibited PDE‐5 enzymatic inhibitory activity, with IC50values of 2.86±0.23, 3.30±1.03, 4.31±0.52, and 4.66±1.13 μM, respectively. The ethanolic extract of the intact roots of EL and EH, and thein vitroroot culture of EH had large amounts of canthin‐6‐one alkaloids (1.50±0.04, 2.12±0.03, and 3.48±0.08 mg/g dry weight, respectively), and showed potent PDE‐5 inhibition. Our findings indicate thatin vitroroot cultures of EH may be used to replace intact plants, and canthin‐6‐one‐9‐O‐β‐D‐glucopyranoside should be further investigated for development as a health supplement.