The inhibitory effect of brown algal phlorotannins on hyaluronidase was evaluated by an in vitro assay. Crude phlorotannins from the brown algae Eisenia bicyclis and Ecklonia kurome had a stronger inhibitory effect than well-known inhibitors such as catechins and sodium cromoglycate. IC50 values of the following six phlorotannins: phloroglucinol, an unknown tetramer, eckol (a trimer), phlorofucofuroeckol A (a pentamer), dieckol and 8,8′-bieckol (hexamers), were 280, 650, >800, 140, 120 and 40 μM, respectively. The IC50 of catechin, epigallocatechin gallate and sodium cromoglycate was 620, 190 and 270 μM, respectively. 8,8′-Bieckol, the strongest HAase inhibitor in this study, acted as a competitive inhibitor with an inhibition constant (Ki) of 35 μM. Acetylation of the phlorotannins markedly decreased their inhibitory potency.