Objective To investigate the in vitro killing effect of halofuginone (Hal) on Echinococcus multilocularis and Trichinella spiralis. Methods E. multilocularis protoscoleces and cysts were isolated from mice infected with E. multilocularis, and muscle larvae, adult worms and newborn larvae of T. spiralis were isolated from mice infected with T. spiralis, followed by in vitro culture in DMEM medium. E. multilocularis protoscolices were treated with Hal at final concentrations of 50, 80, 100 μmol/L, 1% dimethyl sulfoxide (DMSO) and equal volume of medium respectively. After 24, 48 and 72 hours of intervention, the morphology of protoscolices was observed, 0.1% methylene blue staining was used to calculate the mortality of protoscolices in each group, and the half-maximal effective concentration (EC₅₀) of Hal against protoscolices was determined. E. multilocularis cysts were treated with Hal at a final concentration of 100 μmol/L and 1% DMSO respectively, and the ultrastructure of cysts was observed by scanning electron microscopy after 72 hours of intervention. The muscle larvae, adult worms and newborn larvae of T. spiralis were treated with Hal at final concentrations of 50, 80, 100 μmol/L, 1% DMSO and equal volume of medium respectively. The morphology and viability of muscle larvae and adult worms were observed after 24, 48 and 72 hours of intervention, and the morphology and viability of newborn larvae were observed after 96, 120 and 144 hours of intervention. Statistical analysis was performed using GraphPad Prism 9.5, and one-way ANOVA was adopted for data comparison. Results After 48 hours of intervention, the mortality rates of E. multilocularis protoscolices in the 50, 80 and 100 μmol/L Hal groups were (13.05 ± 1.43)%, (57.57 ± 2.57)% and (96.38 ± 2.16)% respectively, all significantly higher than (1.08 ± 0.52)% in the DMSO group (t = 13.587, 37.254, 74.430, all P < 0.01), with an EC₅₀ of 73.96 μmol/L. After 72 hours of intervention with 100 μmol/L Hal, the protoscolices of E. multilocularis showed structural damages including body surface rupture, disordered microvilli arrangement and destroyed suckers under scanning electron microscopy; irregular cracks and pores appeared on the surface of the cyst tegument of E. multilocularis, the germinal layer was extensively exfoliated, and the number of immature protoscolices was significantly reduced. After 72 hours of intervention, the mortality rates of T. spiralis muscle larvae in the 50, 80 and 100 μmol/L Hal groups were (83.59 ± 5.76)%, (87.57 ± 1.52)% and (95.42 ± 1.21)% respectively, all significantly higher than (1.58 ± 0.38)% in the DMSO group (t = 24.590, 94.801, 127.887, all P < 0.01); the mortality rates of adult worms in the 50, 80 and 100 μmol/L Hal groups were (9.32 ± 0.96)%, (36.90 ± 3.08)% and (98.4 ± 1.42)% respectively, all significantly higher than (1.77 ± 0.58)% in the DMSO group (t = 13.201, 19.698, 115.971, all P < 0.01). After 120 hours of intervention, the mortality rates of newborn larvae in the 50, 80 and 100 μmol/L Hal groups were (89.11 ± 1.40)%, (92.32 ± 1.17)% and (95.31 ± 2.31)% respectively, all significantly higher than (1.68 ± 0.16)% in the DMSO group (t = 107.394, 133.302, 70.129, all P < 0.01). After Hal intervention, all T. spiralis muscle larvae, adult worms and newborn larvae showed typical rigid dead morphology without autonomous movement. Conclusion Hal exerts highly efficient anthelmintic effect by destroying the structure of parasites in vitro, which is expected to be a potential drug for the treatment of echinococcosis multilocularis and trichinellosis.