Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality for immunocompromised hosts. We sought to determine the in vitro susceptibility of HCMV reference laboratory strains, clinical isolates and strains with known resistance to currently available anticytomegaloviral drugs to two-drug combinations of the following compounds: ganciclovir, foscarnet, cidofovir and its cyclic congener, cyclic HPMPC (cHPMPC), and lobucavir. Cytotoxicity was determined by Trypan Blue exclusion of cells exposed both when proliferating (non-confluent) and once confluent. Antiviral effect was determined by a plaque-reduction assay in MRC-5 human embryonic lung cells. Drug interactions were determined by median-dose effect analysis with the combination index calculated at 50, 75, 90 and 95% inhibitory concentrations. No drug, either alone or in combination, reached a 50% cytotoxicity concentration in the dose ranges tested. Overall, 252/280 (90.0%) of the two-drug combinations demonstrated additive or synergistic interactive effects towards the panel of HCMV isolates tested. No combination demonstrated antagonism at all inhibitory concentrations to more than one isolate. Interestingly, the clinical isolate tested demonstrated the highest frequency of antagonistic combinations (3/10), as well as marked differences from pan-susceptible laboratory strains. The combinations of ganciclovir + foscarnet and cHPMPC + foscarnet demonstrated additive to synergistic effects against all isolates tested. In vitro combination drug studies could help in the rational choice of therapeutic regimens for use in clinical trials, potentially resulting in decreased toxicity, increased efficacy and delayed onset of drug resistance.