The increasing prevalence of drug-resistant viral infections necessitates the development of novel, safe, and effective antiviral agents. Medicinal mushrooms, known for their therapeutic potential, have gained attention as a promising source of bioactive compounds, particularly β-glucans, for antiviral therapy. This study investigates the antiviral efficacy of β-glucans extracted from Agaricus blazei against Bovine Herpesvirus-1 (BHV-1), a significant pathogen in the cattle industry. β-Glucans, polysaccharides derived from the cell walls of fungi, exhibit immunomodulatory, anti-inflammatory, and antioxidant properties, making them attractive candidates for therapeutic applications. Mushroom-derived β-glucans, characterized by their highly branched structure, have shown antiviral effects against various viruses, including influenza, herpes simplex, and murine norovirus, by enhancing immune responses and potentially inhibiting viral entry or replication. This study outlines a detailed protocol for extracting, purifying, and testing β-glucans from Agaricus blazei for antiviral activity. The protocol includes water and alkaline extraction, purification by dialysis, and ethanol precipitation. The extracted β-glucans are then tested for antiviral activity using a plaque reduction assay on BHV-1-infected HEp-2 cell lines, with plaque reduction observed under a range of treatment conditions. This research highlights the procedural rigor required to assess the potential of mushroom β-glucans as antiviral agents, offering insights into their therapeutic promise in managing viral infections.