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Tetrahymena piriformis belongs to the ciliated protists (ciliates), causing severe economic losses in aquaculture. Chemical drugs currently used usually have toxic side effects, and there is no specific drug against Tetrahymena. Therefore, it is an urgent need to identify new antiparasitic lead compounds. In the present study, the in vitro parasiticidal activity of ethyl acetate (EtOAc) extracts and water extracts from 22 selected traditional Chinese medicines (TCMs) were evaluated against T. piriformis. The EtOAc extract of P. corylifolia turned out to be the most active with the minimum parasiticidal concentration of 100 mg/L within 3 h. Thus, it was separated into 12 fractions by the first-dimensional (D1) normal phase liquid chromatography (NPLC), meanwhile combining with in vitro antiparasitic tests for activity tracking. Subsequently, 8 flavonoids were identified in the active fractions by the second-dimensional (D2) reverse phase liquid chromatography (RPLC) tandem high-resolution mass spectrometry. According to the results, 5 flavonoids were selected for in vitro antiparasitic test, of which isobavachalcone showed the minimum parasiticidal concentration of 3.125 mg/L in 2 h. Bathing treatment of infected guppies with isobavachalcone could significantly reduce the burden of T. piriformis , obtaining a 24-h median effective concentration (24-h EC 50) value of 1.916 mg/L. And the concentration of isobavachalcone causing guppies to die within 24 h is 39 times than that of 24-h EC 50. The results demonstrated that isobavachalcone has the potential to be developed into a novel commercial fish drug against T. piriformis. [Display omitted] • The EtOAc extract of Psoralea corylifolia has activity against Tetrahymena piriformis. • Bavachinin A, Bavachin D, Corylifol A, Neobavaisoflavone and Isobavachalcone were identified. • The acute toxicity and anthelmintic effect of isobavachalcone on Poecilia reticulate were studied. [ABSTRACT FROM AUTHOR]