Patients with clinical manifestations of leishmaniasis, including cutaneous leishmaniasis, have limited treatment options, and existing therapies frequently have significant untoward liabilities. Rapid expansion in the diversity of available cutaneous leishmanicidal chemotypes is the initial step in finding alternative efficacious treatments. To this end, we combined a low-stringency Leishmania major promastigote growth inhibition assay with a structural computational filtering algorithm. After a rigorous assay validation process, we interrogated ∼200,000 unique compounds for L. major promastigote growth inhibition. Using iterative computational filtering of the compounds exhibiting >50% inhibition, we identified 553 structural clusters and 640 compound singletons. Secondary confirmation assays yielded 93 compounds with EC50s ≤ 1 µM, with none of the identified chemotypes being structurally similar to known leishmanicidals and most having favorable in silico predicted bioavailability characteristics. The leishmanicidal activity of a representative subset of 15 chemotypes was confirmed in two independent assay formats, and L. major parasite specificity was demonstrated by assaying against a panel of human cell lines. Thirteen chemotypes inhibited the growth of a L. major axenic amastigote-like population. Murine in vivo efficacy studies using one of the new chemotypes document inhibition of footpad lesion development. These results authenticate that low stringency, large-scale compound screening combined with computational structure filtering can rapidly expand the chemotypes targeting in vitro and in vivo Leishmania growth and viability. Author Summary: Leishmaniasis is a parasitic disease with cutaneous, mucocutaneous and visceral clinical manifestations, depending on the Leishmania spp. and human host. Globally, there are 350 million people at risk of leishmaniasis, but current treatment options rely predominantly on ancient pentavalent antimonials, which have the potential to cause serious systemic toxicity. Our research focuses on the rapid expansion of potential anti-leishmanial compounds that could function as novel chemical structures for future drug development and offer additional therapeutic options to patients with leishmaniasis. We combined high throughput screening methodologies with computational algorithms and multiple confirmatory assay formats to identify and characterize new potent L. major promastigote growth inhibitors, including one that displays in vivo activity without toxicity to human cells. Our use of a large, broadly distributed compound library enabled the identification of these new chemotypes. In addition, since this chemical library is publicly available and annotated, we were able to cross-query archived bioassays and to identify new molecular targets that may be involved in L. major growth and viability as well as identify new protein targets for future leishmanicidal drug discovery. [ABSTRACT FROM AUTHOR]