The main purpose of the present study was undertaken to isolate bioactive substances from microbial secondary metabolites of Streptomyces gilvosporeu AXY-25 and to determine their antiparasitic effect against Ichthyophthirius multifiliis. A pure compound showing strong anti- I. multifiliis activity was isolated from a culture of S. gilvosporeu AXY-25 by using the method of bioassay-guided isolation. The chemical structure of the active compound was confirmed as natamycin (NAT) by spectral analysis (EI-MS, 1H NMR and 13C NMR). An in vitro anti-parasitic assay demonstrated that 100% of theronts were killed with a concentration of 25.0 mg L−1 NAT for 4 h with an effective concentration (EC50) (95% CI) at 10.9 (10.7–11.1) mg L−1. Similiary, all tomonts were killed by a dose of 25.0 mg L−1. An in vivo anti-parasitic assay showed that the number of I. multifiliis trophonts on the surface of hybrid Erythroculter ilishaeformis in the NAT-treated group were markedly lower when compared to the control group (p < 0.05). Mortality in the 25.0 mg L−1 NAT-treated group was 36.7% at day 10. Mortality in the control group due to the I. multifiliis infection was 83.3% by day 10. In addition, the survival rate and reproduction of tomonts in the 25.0 mg L−1NAT treated group were markedly lower than those in the control group (p < 0.05). The results of the acute toxicity of NAT indicated that NAT was safe to use on hybrid E. ilishaeformis, the median lethal concentration (LC50) was 508.6 mg L−1.