The critical consequences of human cytomegalovirus (HCMV) infection in the transplant population and in congenitally infected infants, the limited treatment options for HCMV, and the emergence of resistant mutants during existing therapy fueled the search for new anti-HCMV agents. A pp28-luciferase recombinant HCMV was used as a reporter system for high-throughput screening of HCMV inhibitors. Approximately 400,000 compounds from existing libraries were screened. Subsequent validation assays using resynthesized compounds, several virus strains and detailed virology assays resulted in the identification of five structurally unique and selective HCMV inhibitors, active at sub to low ┬ÁM concentrations. Further characterization revealed that each compound inhibited a specific stage of HCMV replication. Only one compound was also active against herpes simplex virus. Drug combination studies revealed that all five compounds were additive with ganciclovir or letermovir. Future studies will determine the potential for development of these new compounds as anti-HCMV drugs and their use as probes to study virus-host interaction.