Ichthyophthirius multifiliis (Ich), a fish ectoparasite, comprises an important challenge in the aquaculture industry. In this study, a steroidal glycoside, cynatratoside-C, isolated from Cynanchum atratum roots by bioassay-guided fractionation was used to treat I. multifiliis. The cynatratoside-C at 0.25 mg/L demonstrated a 100% mortality of I. multifiliis in vitro after 5 h exposure. The 5 h median effective concentration (EC50) of cynatratoside-C to nonencysted tomonts was 0.083 mg/L. In addition, cynatratoside-C at concentrations of 0.125 and 0.06 mg/L could completely terminate the reproduction of encysted tomonts. The cynatratoside-C at 2 mg/L could cure the infected grass carp within 48 h. The exact mechanism of cynatratoside-C for killing I. multifiliis is unknown, but it manifests itself microscopically through loss of membrane integrity of nonencysted tomonts or through releasing immature theronts from encysted tomonts. The immature theronts finally died before infecting fish. On the basis of these results, cynatratoside-C could be used as a natural anti-I. multifiliis agent.