It has been generally accepted that hydantoin [5-(3,4-dichlorophenyl)methylhydantoin] exerts its anti-enterovirus activity by solely inhibiting viral assembly. However, we here show that hydantoin inhibits enteroviral RNA synthesis as well as subgenomic replication in a dose-dependent manner. We demonstrate that inhibition of RNA synthesis is the predominant mechanism of action at relatively high concentrations of hydantoin. However, at lower concentrations inhibition of viral morphogenesis is the main mechanism of action. Thus, hydantoin inhibits enteroviral replication by two distinct mechanisms.