By the method of co-culture, this paper studied the inhibitory effect of Surgassum thunbergii culture filtrate, fresh tissue, dry powder, and aqueous extract on the growth of Heterosigma akashiwo and Skeletonema costatum. An isolated co-culture system was built, which confirmed the existence of allelochemicals and precluded the growth inhibition by direct contact. The results showed that the fresh tissue, dry powder, and aqueous extract of S. thunbergii could significantly inhibit the growth of H. akashiwo and S. costatum, and had lethal effect at relatively higher concentrations. The culture filtrate of S. thunbergii had no inhibitory effect on H. akashiwo, but could significantly inhibit the growth of S. costatum under initial or semi-continuous filtrate addition.