Human cytomegalovirus (HCMV) infection in utero can lead to congenital sensory neural hearing loss and mental retardation. Reactivation or primary infection can increase the morbidity and mortality in immune suppressed transplant recipients and AIDS patients. The current standard of care for HCMV disease is nucleoside analogs, which can be nephrotoxic. In addition resistance to current treatments is becoming increasingly common. In an effort to develop novel CMV treatments, we tested the effectiveness of the D-form of a novel heparan sulfate binding peptide, p5RD, at reducing infection of ganciclovir (GCV) resistant HCMVs in vitro and MCMV in vivo. HCMV infection was reduced by greater than 90% when cells were pretreated with p5RD. Because p5RD acts by a mechanism unrelated to those used by current antivirals, it was effective at reducing GCV resistant HCMVs by 85%. We show that p5RD is resistant to common proteases and serum inactivation, which likely contributed to its ability to significantly reduced infection of peritoneal exudate cells and viral loads in the spleen and the lungs in vivo. The ability of p5RD to reduce HCMV infectivity in vitro including GCV resistant HCMVs and MCMV infection in vivo suggests that this peptide could be a novel anti-CMV therapeutic.