Four β-lactone- γ-lactam proteasome inhibitors of natural origin were tested for their trypanocidal activities in vitro using culture-adapted bloodstream forms of Trypanosoma brucei. All four compounds displayed activities in the nanomolar range. The most trypanocidal compounds with 50% growth inhibition (GI(50)) values of around 3 nM were the bromine and iodine analogues of salinosporamide A, a potent proteasome inhibitor produced by the marine actinomycete Salinispora tropica. In general, trypanosomes were more susceptible to the compounds than were human HL-60 cells. The data support the potential of β-lactone- γ-lactam proteasome inhibitors for rational anti-trypanosomal drug development.