ABSTRACT Human cytomegalovirus (HCMV) is a major cause of disease in immunocompromised individuals and the most common cause of congenital infection and neurosensorial disease. The expanding target populations for HCMV antiviral treatment along with the limitations of the currently available HCMV DNA polymerase inhibitors underscore the need for new antiviral agents with alternative modes of action. The antimalarial artemisinin derivative artesunate was shown to inhibit HCMV in vitro yet has demonstrated limited antiviral efficacy in vivo , prompting our search for more potent anti-HCMV artemisinin derivatives. Here we show that the innovative artemisinin derivative artemisone, which has been screened for its activity against malaria parasites in human clinical studies, is a potent and noncytotoxic inhibitor of HCMV. Artemisone exhibited an antiviral efficacy comparable to that of ganciclovir (50% effective concentration, 1.20 ± 0.46 μM) in human foreskin fibroblasts, with enhanced relative potency in lung fibroblasts and epithelial cells. Significantly, the antiviral efficacy of artemisone was consistently ≥10-fold superior to that of artesunate in all cells. Artemisone effectively inhibited both laboratory-adapted and low-passage-number clinical strains, as well as drug-resistant HCMV strains. By using quantitative viral kinetics and gene expression studies, we show that artemisone is a reversible inhibitor targeting an earlier phase of the viral replication cycle than ganciclovir. Importantly, artemisone most effectively inhibited HCMV infection ex vivo in a clinically relevant multicellular model of integral human placental tissues maintained in organ culture. Our promising findings encourage preclinical and clinical studies of artemisone as a new inhibitor against HCMV.