Herpes simplex virus type 1 (HSV-1) is a double-stranded DNA virus that infects most of the human population. We previously found that cellular topoisomerase I (TOP1) associates with the HSV-1 genome throughout infection. TOP1 relieves topological stress on DNA to enable and regulate transcription, DNA replication, and DNA repair. We hypothesized that TOP1 contributes to these key viral processes because HSV-1 does not encode a topoisomerase. We found that TOP1 inhibition with camptothecin (CPT) or β-Lapachone results in a significant reduction in viral yield. The effects of CPT are reversible and occur in a dose-dependent manner, independent of strain and cell type. CPT treatment results in a decrease in viral gene expression and DNA replication, with replication defects observed at all concentrations tested. Defects in viral gene expression are not restricted to a specific gene class. However, the timing and extent of the observed defects are dose dependent. We also demonstrate that if CPT is added to cells after the onset of viral DNA replication, subsequent rounds of replication are inhibited, indicating that the replication defect observed is not simply the result of prior steps in the infectious cycle being blocked. Together, these results demonstrate that CPT is a potent inhibitor of HSV-1 infection, supporting the hypothesis that TOP1 is active on HSV-1 genomes during infection.IMPORTANCEHSV-1 is a common human pathogen. It utilizes both viral and cellular factors to facilitate infection. We previously characterized the proteins that associate with HSV-1 DNA throughout infection. One of these proteins, which has been largely unexplored in the context of HSV-1 infection, is TOP1. TOP1 functions to relieve topological stress to regulate transcription, DNA replication, and other processes that involve DNA cleavage and unwinding. Our findings support a role for TOP1 in HSV-1 DNA replication and gene expression and highlight the potential to target TOP1 activity or interactions for antiviral therapy.