The present study was conducted to evaluate the anti-parasitic activity of a pure compound from Streptomyces sp. HL-2-14 against fish parasite Ichthyophthirius multifiliis, and elucidate its chemical structure. By electron ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance spectrum (1H NMR and 13C NMR), the compound was identified as amphotericin B (AmB). The in vitro trials revealed that AmB can effectively kill the theronts and tomonts of I. multifiliis with the median lethal concentration (LC50) of 0·8 mg L−1 at 30 min for the theronts and 4·3 mg L−1 at 2 h for the tomonts, respectively. AmB at 5 mg L−1 significantly reduced I. multifiliis infectivity prevalence and intensity on grass carp (Ctenopharyngodon idella), and consequently decreased fish mortality, from 100% in control group to 30% in treated group. The 72 h acute toxicity (LC50) of AmB on grass carp was 20·6 mg L−1, but fish mortality was occurred when exposure to 13·0 mg L−1. These results indicated that AmB was effective in the therapy of I. multifiliis infection, but the safety concentration margin is relatively narrow. Further efforts aiming to decrease the toxicity and improve the therapeutic profile remain to be needed.