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Abstract Background Developing new antibabesial drugs with a low toxic effect to the animal and with no resistance from Babesia parasites is in urgent demand. In this concern, the antimalarial, anticancer and antioxidant effect of thymoquinone (TQ), a phytochemical compound found in the plant Nigella sativa, has been reported. Therefore, in the present study, the antibabesial effect of this compound was evaluated on the growth of piroplasm parasites. Results Significant inhibition (P < 0.05) of the in vitro growth of piroplasm parasites were observed after treatment by TQ with IC50 values of 35.41 ± 3.60, 7.35 ± 0.17, 0.28 ± 0.016, 74.05 ± 4.55 and 67.33 ± 0.94 μM for Babesia bovis, Babesia bigemina, Babesia divergens, Theileria equi and Babesia caballi, respectively. The in vitro inhibitory effect of TQ was significantly enhanced (P < 0.05) when used in combination with either diminazene aceturate on bovine Babesia and equine Babesia and Theileria cultures. In B. microti-infected mice, oral and intraperitoneal administrations of TQ showed significant (P < 0.05) inhibition of parasite growth at a dose of 70 mg/kg and 50 mg/kg, respectively, compared to the control group. Conclusions The obtained results indicate that thymoquinone might be a promising medicinal compound for use in the treatment of animal piroplasmosis.