Dactylogyrus intermedius is a significant monogenean parasite on the gills of cyprinid fishes and can cause serious problem in fish aquaculture. In the present study, bioassay-guided fractionation was employed to identify the active compounds from Fructus Arctii against D. intermedius. Five solvents (petroleum ether, chloroform, ethyl acetate, ethanol, and water) were applied for the extraction of Fructus Arctii. Among them, only the chloroform extract exhibited promising anthelmintic efficacy and therefore, subjected to the further isolation and purification using various chromatographic techniques. Two compounds showing potent activity were obtained and identified by spectral data (infrared, nuclear magnetic resonance, and mass spectrometry) as: arctigenin (1) and arctiin (2). They were found to be significantly effective against D. intermedius with median effective concentration (EC(50)) values of 0.62 and 3.55 mg L(-1), respectively. Arctigenin exhibited higher activity as compared with the positive control mebendazole with an EC(50) value of 1.25 mg L(-1). The 48-h acute toxicity tests (LC(50)) of arctigenin and arctiin were found to be 8.47 and 14.14 mg L(-1) for goldfish, respectively. These results provided evidence that the studied plant extract, as well as the isolated compounds, might be potential sources of new antiparasitic drug for the control of Dactylogyrus.