Enzymes of the M32 family are Zn-dependent metallocarboxypeptidases (MCPs) widely distributed among prokaryotic organisms and just a few eukaryotes including Trypanosoma brucei and Trypanosoma cruzi, the causative agents of sleeping sickness and Chagas disease, respectively. These enzymes are absent in humans and several functions have been proposed for trypanosomatid M32 MCPs. However, no synthetic inhibitors have been reported so far for these enzymes. Here, we present the identification of a set of inhibitors for TcMCP-1 and TbMCP-1 (two trypanosomatid M32 enzymes sharing 71% protein sequence identity) from the GlaxoSmithKline HAT and CHAGAS chemical boxes; two collections grouping 404 compounds with high antiparasitic potency, drug-likeness, structural diversity and scientific novelty. For this purpose, we adapted continuous fluorescent enzymatic assays to a medium-throughput format and carried out the screening of both collections, followed by the construction of dose-response curves for the most promising hits. As a result, 30 micromolar-range inhibitors were discovered for one or both enzymes. The best hit, TCMDC-143620, showed sub-micromolar affinity for TcMCP-1, inhibited TbMCP-1 in the low micromolar range and was inactive against angiotensin I-converting enzyme (ACE), a potential mammalian off-target structurally related to M32 MCPs. This is the first inhibitor reported for this family of MCPs and considering its potency and specificity, TCMDC-143620 seems to be a promissory starting point to develop more specific and potent chemical tools targeting M32 MCPs from trypanosomatid parasites.