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The use of plant-derived natural products for the treatment of tropical parasitic diseases often has ethnopharmacological origins. As such, plants grown in temperate regions remain largely untested for novel anti-parasitic activities. We describe here a screen of the PhytoQuest Phytopure library, a novel source comprising over 600 purified compounds from temperate zone plants, against in vitro culture systems for <i>Plasmodium falciparum</i>, <i>Leishmania mexicana</i>, <i>Trypanosoma evansi</i> and <i>T. brucei</i>. Initial screen revealed 6, 65, 15 and 18 compounds, respectively, that decreased each parasite’s growth by at least 50% at 1–2 µM concentration. These initial hits were validated in concentration-response assays against the parasite and the human HepG2 cell line, identifying hits with EC₅₀ < 1 μM and a selectivity index of >10. Two sesquiterpene glycosides were identified against <i>P. falciparum</i>, four sterols against <i>L. mexicana</i>, and five compounds of various scaffolds against <i>T. brucei</i> and <i>T. evansi</i>. An <i>L. mexicana</i> resistant line was generated for the sterol 700022, which was found to have cross-resistance to the anti-leishmanial drug miltefosine as well as to the other leishmanicidal sterols. This study highlights the potential of a temperate plant secondary metabolites as a novel source of natural products against tropical parasitic diseases.