The bovine herpesvirus type I (BHV-1) is a significant pathogen that poses a threat to the healthy development of the cattle industry and has a global prevalence. Paeonol is a phenolic constituent extracted from the dried root bark of peony in the buttercup family. Although paeonol has anti-inflammatory and antioxidant effects, its antiviral capacity remains unclear. Here, we conducted a cytotoxicity assay to screen the safe concentration of the paeonol using bovine turbinate (BT) cells as a model. Antiviral studies showed that paeonol inhibited BHV-1 gB gene and VP8 protein expression, and reduced cytopathic effect and viral titer. Furthermore, paeonol also demonstrated a potent effect on BHV-1 replication with an EC