Dactylogyrus intermedius is one of the most pathogenic monogenean parasites on the gills of captive fish and can cause serious problem in aquaculture. To attempt controlling this parasite and explore novel potential antiparasitic agents, the present study was designed to investigate the anthelmintic activity of Dioscorea zingiberensis C. H. Wright against D. intermedius in goldfish under in vivo conditions. Bioactivity-guided fractionation and isolation of the compounds responsible for anthelmintic activity was carried out with the ethanolic extract yielding two bioactive compounds. Using MS, (1)H NMR, (13)C NMR spectroscopic analyses, the two compounds were identified as trillin and gracillin. The results of in vivo anthelmintic efficacy assay showed that the 48-h median effective concentrations (EC(50)) are 26.48 mg L(-1) for trillin and 0.18 mg L(-1) for gracillin. The 48-h acute toxicity tests (LD(50)) of trillin and gracillin were found to be 73.11 and 1.40 mg L(-1) for goldfish, respectively. The resulting therapeutic indices for the two active compounds are 2.76 and 7.78, respectively. These data confirmed that both trillin and gracillin are effective against D. intermedius, and the gracillin exhibits more interesting perspectives for the development of a candidate antiparasitic agent.