Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus for which there is no vaccine or cure. This viral infection, once acquired, is life-long, residing latently in hematopoietic cells. However, latently infected individuals with weakened immune systems often undergo HCMV reactivation, which can cause serious complications in immunosuppressed and immunocompromised patients. Current anti-viral therapies target late stages of viral replication, and are often met with therapeutic resistance, necessitating the development of novel therapeutics. In this current study, we identified a naturally-occurring flavonoid compound, deguelin, which inhibits HCMV lytic replication. Our findings reveal that nanomolar concentrations of deguelin significantly suppress the production of the infectious virus. Further, we show that deguelin inhibits the lytic cycle during the phase of the replication cycle consistent with early (E) gene and protein expression. Importantly, our data reveal that deguelin inhibits replication of a ganciclovir-resistant strain of HCMV. Together, our findings identify a novel, naturally occurring compound that may prove useful in the treatment of HCMV replication.