Abstract The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by toxicity and emergent resistance. Furthermore, our understanding of drug mode-of-action and potential routes to resistance is limited. Forward genetic approaches have revolutionised our understanding of drug mode-of-action in the related kinetoplastid parasite, Trypanosoma brucei. Therefore, we screened our genome-scale T. brucei RNAi library in the current anti-leishmanial drugs, sodium stibogluconate (antimonial), paromomycin, miltefosine and amphotericin-B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource. Follow-up analyses revealed the antimonial selectivity of the aquaglyceroporin, TbAQP3. A lysosomal major facilitator superfamily transporter contributes to paromomycin/aminoglycoside efficacy. The vesicle-associated membrane protein, TbVAMP7B, and a flippase contribute to amphotericin-B and miltefosine action, and are potential cross-resistance determinants. Finally, multiple phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania miltefosine transporter, a putative β-subunit/CDC50 co-factor, and additional membrane-associated hits, affect amphotericin-B efficacy, providing new insights into mechanisms of drug uptake and action. The findings from this orthology-based chemogenomic profiling approach substantially advance our understanding of anti-leishmanial drug action and potential resistance mechanisms, and should facilitate the development of improved therapies, as well as surveillance for drug-resistant parasites. Importance Leishmaniasis is a devastating disease caused by the Leishmania parasites and is endemic to a wide swathe of the tropics and sub-tropics. While there are drugs available for the treatment of leishmaniasis, these suffer from various challenges, including the spread of drug resistance. Our understanding of anti-leishmanial drug action and the modes of drug resistance in Leishmania is limited. The development of genetic screening tools in the related parasite, Trypanosoma brucei, has revolutionised our understanding of these processes in this parasite. Therefore, we applied these tools to the anti-leishmanial drugs, identifying T. brucei orthologues of known Leishmania proteins that drive drug uptake, as well as a panel of novel proteins not previously associated with anti-leishmanial drug action. Our findings substantially advance our understanding of anti-leishmanial mode-of-action and provide a valuable starting point for further research.