The genome packaging of human cytomegalovirus (HCMV) requires a divalent metal-dependent endonuclease activity localized to the C-terminus of pUL89 (pUL89-C), which is reminiscent of RNase H-like enzymes in active site structure and catalytic mechanism. Our previous work has shown that metal-binding small molecules can effectively inhibit pUL89-C while conferring significant antiviral activities. In this report we generated a collection of 43 metal-binding small molecules by repurposing analogs of the 6-arylthio-3-hydroxypyrimidine-2,4-dione chemotype previously synthesized for targeting HIV-1 RNase H, and by chemically synthesizing new N-1 analogs. The analogs were subjected to two parallel screening assays: the pUL89-C biochemical assay and the HCMV antiviral assay. Compounds with significant inhibition from each assay were further tested in a dose-response fashion. Single dose cell viability and PAMPA cell permeability were also conducted and considered in selecting compounds for the dose-response antiviral testing. These assays identified a few analogs displaying low μM inhibition against pUL89-C in the biochemical assay and HCMV replication in the antiviral assay. The target engagement was further evaluated via a thermal shift assay using recombinant pUL89-C and molecular docking. Overall, our current work identified novel inhibitors of pUL89-C with significant antiviral activities and further supports targeting pUL89-C with metal-binding small molecules as an antiviral approach against HCMV.