Natural products are an inexhaustible source of compounds with promising pharmacological activities, including antiviral action. In the present study, the antiviral potential of polysaccharide-peptide (PLS) and an extracted β-glucan from Agaricus brasiliensis were investigated in the replication of bovine herpesvirus 1 (BoHV-1) in HEp-2 cell cultures. The cytotoxicity (CC50) was assayed by the MTT method and the antiviral activity (IC50) was estimated by the plaque reduction assay. To study the possible mode of action of PLS and β-glucan, the following protocols were performed: the virucidal assay, adsorption assay and the time-of-addition assay. The PLS presented a selectivity index (SI) higher than 12.50 and β-glucan 9.19. The antiviral inhibition (67.9%) in cells treated with PLS during virus infection was higher than that in cells treated prior to or post infection. The β-glucan presented high inhibition of virus replication by plaque assay (83.2%) and by immunofluorescence assay (63.8%). Although the mechanism has yet to be defined, we suggest that PLS and β-glucan inhibited BoHV-1 replication by interfering with the early events of viral penetration. Additional studies are required for a better understanding of the mechanism of action of PLS and β-glucan.