Cutaneous and mucosal human papillomaviruses (HPVs) are implicated in the development of various cancers. To date, there are no HPV-specific antivirals, and vaccines designed for mucosal HPV types are ineffective against cutaneous HPV variants and ongoing infections. These facts, along with the increasing prevalence of specific types of HPV-positive cancers, underscore the unmet need for HPV antiviral drugs. In this study, we performed a high-throughput screening using the National Cancer Institute Diversity Set VI library of small-molecule compounds and identified 5,7-dimethoxy-2-pyridin-3-ylchromen-4-one (NSC51349) as a potent inhibitor of oncogenic cutaneous HPV5 replication in U2OS cells. Further analysis showed that NSC51349 specifically inhibits replication of the HPV5 genome in human primary keratinocytes (IC50 ≈ 4 µM) without detrimental effects on cell cycle, viability, or differentiation. In addition, this compound efficiently inhibits replication of the other cutaneous HPV types 8 and 38, and Macaca fascicularis PV type 1 genomes. The mechanism of NSC51349-mediated inhibition involves the suppression of the viral protein E2 transcriptional activity. Molecular docking predicted that NSC51349 interacts with the HPV5 E2 protein by binding to the threonine 202/proline 203 and threonine 473/glutamate 474 amino acid residues, located at the junction of the transactivation domain and hinge region, and the DNA-binding domain, respectively. Our study suggests that NSC51349 is a promising lead compound for further development of cutaneous HPV antiviral drugs.IMPORTANCEHuman papillomaviruses (HPVs) are linked to various cancers of the skin and mucous membranes. While vaccines exist for some mucosal HPV types, they are ineffective against skin-infecting variants and cannot treat existing infections. This highlights the urgent need for HPV-specific antiviral drugs. In this study, we identified a promising lead compound, 5,7-dimethoxy-2-pyridin-3-ylchromen-4-one (NSC51349), through high-throughput screening of the Diversity Set VI library of small molecules. NSC51349 inhibits the replication of HPV5, a cancer-associated skin virus, in human cells without affecting cell viability, growth, or differentiation. It also inhibits the replication of other cutaneous HPV types, including HPV8 and HPV38, further supporting its broad potential. NSC51349 targets the viral protein E2, which is crucial for HPV replication. By binding to specific regions of E2, it interferes with its transcriptional activity, halting viral replication and the expression of viral oncogenes. This study introduces NSC51349 as a strong candidate for antiviral drug development.