Phytochemicals are natural compounds found in plants that have potential health benefits such as antioxidants, anti-inflammatory and anti-cancer properties, and immune reinforcement.Polygonum cuspidatumSieb. et Zucc. is a source rich in resveratrol, traditionally consumed as an infusion. In this study,P. cuspidatumroot extraction conditions were optimized to increase antioxidant capacity (DPPH, ABTS+), extraction yield, resveratrol concentration, and total polyphenolic compounds (TPC) via ultrasonic-assisted extraction using a Box–Behnken design (BBD). The biological activities of the optimized extract and the infusion were compared. The optimized extract was obtained using a solvent/root powder ratio of 4, 60% ethanol concentration, and 60% ultrasonic power. The optimized extract showed higher biological activities than the infusion. The optimized extract contained 16.6 mg mL−1resveratrol, high antioxidant activities (135.1 µg TE mL−1for DPPH, and 230.4 µg TE mL−1for ABTS+), TPC (33.2 mg GAE mL−1), and extraction yield of 12.4%. The EC50value (effective concentration 50) of the optimized extract was 0.194 µg mL−1, which revealed high cytotoxic activity against the Caco-2 cell line. The optimized extract could be used to develop functional beverages with high antioxidant capacity, antioxidants for edible oils, functional foods, and cosmetics.