Six dietary isothiocyanates, allyl-isothiocyanate, benzyl-isothiocyanate, phenylethyl-isothiocyanate, sulforaphane, erucin, and iberin, were tested for their trypanocidal activities in vitro using culture-adapted bloodstream forms of Trypanosoma brucei. All isothiocyanates showed a dose-dependent effect on the growth of trypanosomes. Five compounds displayed MIC values of 10 μM and GI values of around 1.5 μM, while allyl-isothiocyanate exhibited values of 100 and 11 μM, respectively. The compounds showed similar cytotoxic activities against human HL-60 cells with GI values of 1-4 μM and MIC values of 10-100 μM. Short-term experiments revealed that, with the exception of allyl-isothiocyanate, isothiocyanates at a concentration of 10 μM kill trypanosomes within 1-4 h of incubation. In contrast, HL-60 cells were not affected by any of the compounds in short-term incubation experiments. Sulforaphane, the most intensively studied isothiocyanate, was also investigated for its in vivo trypanocidal activity. However, administration of 50 mg/kg sulforaphane orally or intraperitoneally for four days had no effect on the parasitaemia in mice infected with T. brucei compared to control animals treated with vehicle alone.