A wide range of antiviral drugs is currently available; however, drug-resistant viruses have begun to emerge and represent a potential public health risk. Here, we explored the use of compounds that inhibit or interfere with the action of essential host factors to prevent virus replication. In particular, we focused on the cyclin-dependent kinase 9 (CDK9) inhibitor, FIT-039, which suppressed replication of a broad spectrum of DNA viruses through inhibition of mRNA transcription. Specifically, FIT-039 inhibited replication of herpes simplex virus 1 (HSV-1), HSV-2, human adenovirus, and human cytomegalovirus in cultured cells, and topical application of FIT-039 ointment suppressed skin legion formation in a murine HSV-1 infection model. FIT-039 did not affect cell cycle progression or cellular proliferation in host cells. Compared with the general CDK inhibitor flavopiridol, transcriptome analyses of FIT-039–treated cells revealed that FIT-039 specifically inhibited CDK9. Given at concentrations above the inhibitory concentration, FIT-039 did not have a cytotoxic effect on mammalian cells. Importantly, administration of FIT-039 ameliorated the severity of skin lesion formation in mice infected with an acyclovir-resistant HSV-1, without noticeable adverse effects. Together, these data indicate that FIT-039 has potential as an antiviral agent for clinical therapeutics.