Since the eradication of smallpox, zoonotic poxviruses, such as the mpox virus (MPXV), continue to pose a threat to public health. Identifying drugs that reduce poxvirus infection and replication, as well as understanding their molecular mechanisms, is essential for epidemic control. Polo-like kinase 1 (PLK1) has been shown to facilitate vaccinia virus (VACV) infection and replication. This study confirms the effects of the PLK1 inhibitors HMN-214 and ON-01910 on VACV replication in A549 cells. Both viral titers and DNA loads were significantly reduced in treated cells after infection. Additionally, ON-01910 demonstrated broad-spectrum antiviral activity against the lumpy skin disease virus (LSDV) and the infectious bovine rhinotracheitis virus (IBRV) in vitro. PLK1 knockdown in A549 cells also led to a reduction in VACV protein expression, viral titers, and DNA levels. Further analysis showed that VACV infection leads to the accumulation of PLK1 near viral factories. However, despite its strong in vitro effects, ON-01910 did not significantly reduce VACV replication in mice. These findings highlight the critical role of PLK1 in VACV replication and its potential as a target for antiviral therapy against orthopoxviruses.