Access to large phenotypic screens has enabled the discovery of novel anti-infective compounds. Translating these compounds into new drugs faces a number of challenges. Finding the mode-of-action (MoA) can help in focusing the efforts to develop the most promising leads. Using a medium-throughput method, we performed untargeted metabolomics analyses of Trypanosoma brucei, a kinetoplastid parasite which causes a neglected tropical disease known as sleeping sickness. This allowed us to investigate the mode of action of over 20 potent trypanocidal compounds from the Pathogen Box (available from Medicines for Malaria Venture). Over 500 metabolite features were identified in this study and mapped to the predicted metabolic network. This approach identified the metabolic pathways targeted by the most potent compounds which spanned nucleotide metabolism, lipid metabolism, co-factor synthesis and redox metabolism. Lysophospholipid metabolism, glutamate metabolism, purine salvage and S-adenosyl-methionine and polyamine pathways were found to be specifically hit. Interestingly, multivariate analyses showed that compounds with similar chemical structures did not always have similar biochemical activities. This study showed that a simple metabolomics assay can rapidly reveal the MoA of newly discovered anti-infective compounds. This information can be used for prioritising compounds in the optimization pipeline and help in designing combination therapies that target discrete pathways to overcome emerging drug-resistance.