Previously we established reporter cell lines for human cytomegalovirus (HCMV) and varicella zoster virus (VZV) and identified several antiviral compounds against these viruses using the reporter cells. In this study, we found that one of the identified anti-HCMV compounds, a thienylcarboxamide derivative (coded as 133G4), was effective against not only HCMV but also VZV. The following findings indicate that 133G4 inhibits the activation of early gene promoters by HCMV IE2 and VZV IE62: i) 133G4 decreased the expression of HCMV early and late genes but not that of HCMV IE1/IE2 in HCMV-infected cells, ii) 133G4 inhibited the activation of several HCMV early gene promoters of transiently-transfected plasmids in HCMV-infected cells, and iii) in transient transfection assays, 133G4 decreased the activation of HCMV (or VZV) early gene promoters by HCMV IE2 (or VZV IE62) in the absence of other viral protein expression. The inhibition of early gene activation was observed in the human and African green monkey cell lines but not in the rodent cell lines, and the compound was not effective against murine CMV. In addition, VZV IE62 activated HCMV early promoters, and 133G4 still inhibited such promoter activation. Therefore, we hypothesized that 133G4 targets a cellular factor used commonly in activation of human herpesvirus promoters and examined whether 133G4 affects the functions of cellular proteins USF1, TBP, Med25 and EAP, the involvement of which in VZV IE62-dependent viral gene activation has been well characterized. Our experimental results using one-hybrid and bimolecular fluorescence complementation assays demonstrated that 133G4 did not inhibit the recruitment of USF1 or TBP to their binding sites, nor inhibited the direct interactions of VZV IE62 with Med25 and EAP. Thus, 133G4 is a unique anti-VZV and -HCMV compound, which warrants further studies to find out its inhibitory mechanism.