Treatment of human cytomegalovirus (HCMV) infections with any of the currently available antiviral agents is frequently associated with the occurrence of severe complications, seriously threatening the successful outcome of treatment. Therefore, the development of novel antiviral strategies is a challenging goal of current investigations. Previously, we reported that artesunate (ART) is an effective, non-cytotoxic inhibitor of HCMV in vitro. Here, we demonstrate that the efficacy of the antiviral effect of ART is augmented by co-treatment of HCMV-infected fibroblasts with ferrous iron, i.e. Ferrosanol, and/or the iron transfer-mediating molecule holo-transferrin. This could alleviate the HCMV-induced modulation of cell surface expression of adhesion molecule Thy-1, suggesting that ART might be able to prevent pro-inflammatory effects of infection. The iron-enhanced, antiviral effect of ART could also be demonstrated in cultured cells infected with rat cytomegalovirus. Experiments using the RCMV/rat model showed that both the viral DNA load and virus titers in the salivary glands from infected rats were significantly reduced upon treatment with ART. Furthermore, an additive antiviral effect for ART together with each one of conventional anti-HCMV drugs, i.e. ganciclovir, cidofovir or foscarnet, was detected in HCMV-infected fibroblasts. These findings might open new perspectives regarding the use of ART in clinical trials.