This study investigated the antiviral potential of fractions and eupomatenoid-5 obtained from Piper regnelli (Miq.) C. DC., Piperaceae, leaves against bovine herpesvirus-1 (BHV-1) and poliovirus. VERO cell monolayers in 96-well cell culture plates were infected with BHV-1 or poliovirus and incubated in the presence and absence of samples for 48 h. The cells were then fixed and stained with sulforhodamine B, and the virus-induced cytopathic effect was measured in a 96-well plate reader at 530 nm. Cytotoxicity was assessed by incubating the cell monolayers with samples for 72 h. The hexane, chloroform, chloroform/ethyl acetate (19:1), and chloroform/ethyl acetate (9:1) fractions showed activity against BHV-1. The chloroform, chloroform/ethyl acetate (19:1), chloroform/ethyl acetate (9:1), chloroform/ethyl acetate (1:1), and ethyl acetate fractions were active against poliovirus. The chloroform/ethyl acetate (9:1) fraction presented the best selectivity index for both viruses. The present study reports the antiviral activity of the extract and fractions of P. regnelli leaves.