Eurycoma longifolia (EL) and Eurycoma 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, beta-carboline alkaloids, and quassinoids were also screened for PDE-5 inhibitory activity. We developed in vitro root 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-beta-D-glucopyranoside, 9-methoxycanthin-6-one, canthin-6-one, and 9-hydroxycanthin-6-one, exhibited PDE-5 enzymatic inhibitory activity, with IC(50) values of 2.86+/-0.23, 3.30+/-1.03, 4.31+/-0.52, and 4.66+/-1.13 muM, respectively. The ethanolic extract of the intact roots of EL and EH, and the in vitro root 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 that in vitro root cultures of EH may be used to replace intact plants, and canthin-6-one-9-O-beta-D-glucopyranoside should be further investigated for development as a health supplement. CI - (c) 2022 Wiley-VHCA AG, Zurich, Switzerland.