An extract of whole plants of Pycnanthemum flexuosum showed an inhibitory effect on hyaluronidase activity. From an 80% acetone extract of aerial parts, 3-[(3E)-4-phenylbut-3-enoylamino]propionic acid, 3-O-β-d-glucuronopyranosyl-echinocystic acid 28-O-β-d-xylopyranosyl-(1→3)-[3,4-diacetyl-β-d-xylopyranosyl-(1→4)]-α-l-rhamnopyranosyl-(1→2)-α-l-arabinopyranosyl ester, vanillic acid 1-O-[(5-O-syringoyl)-β-d-apiofuranosyl]-(1→2)-β-d-glucopyranoside, and (4S,5R)-4-hydroxy-5-phenyl-tetrahydrofuran-2-one were isolated together with 30 known compounds. Six known compounds were isolated from an 80% acetone extract of roots, and eritrichin was revealed as a hyaluronidase inhibitor in P. flexuosum.