The present study aims to evaluate the antiparasitic activity of active components from Costus speciosus against Ichthyophthirius multifiliis. Bioassay-guided fractionation was employed to identify active compounds from C. speciosus yielding 2 bioactive compounds: Gracillin and Zingibernsis newsaponin. In-vitro assays revealed that Gracillin and Zingibernsis newsaponin could be 100% effective against I. multifiliis at concentrations of 0·8 and 4·5 mg L−1, with median effective concentration (EC50) values of 0·53 and 3·2 mg L−1, respectively. All protomonts and encysted tomonts were killed when the concentrations of Gracillin and Zingibernsis newsaponin were 1·0 and 5·0 mg L−1. In-vivo experiments demonstrated that fish treated with Gracillin and Zingibernsis newsaponin at concentrations of 1·0 and 5·0 mg L−1 carried significantly fewer parasites than the control (P<0·05). Mortality of fish did not occur in the treatment group (Zingibernsis newsaponin at 5·0 mg L−1) during the trial, although 100% of untreated fish died. Acute toxicities (LD50) of Gracillin and Zingibernsis newsaponin for grass carp were 1·64 and 20·7 mg L−1, respectively. These results provided evidence that the 2 compounds can be selected as lead compounds for the development of new drugs against I. multifiliis.