Apicomplexans are widespread parasites of humans and other animals, and include the causative agents of malaria (Plasmodium species) and toxoplasmosis (Toxoplasma gondii). Existing anti-apicomplexan therapies are beset with issues around drug resistance and toxicity, and new treatment options are needed. The mitochondrial electron transport chain (ETC) is one of the few processes that has been validated as a drug target in apicomplexans. To identify new inhibitors of the apicomplexan ETC, we developed a Seahorse XFe96 flux analyzer approach to screen the 400 compounds contained within the Medicines for Malaria Venture 'Pathogen Box' for ETC inhibition. We identified six chemically diverse, on-target inhibitors of the ETC in T. gondii, at least four of which also target the ETC of Plasmodium falciparum. Two of the identified compounds (MMV024937 and MMV688853) represent novel ETC inhibitor chemotypes. MMV688853 belongs to a compound class, the aminopyrazole carboxamides, that were shown previously to target a kinase with a key role in parasite invasion of host cells. Our data therefore reveal that MMV688853 has dual targets in apicomplexans. We further developed our approach to pinpoint the molecular targets of these inhibitors, demonstrating that all target Complex III of the ETC, with MMV688853 targeting the ubiquinone reduction (Qi) site of the complex. Most of the compounds we identified remain effective inhibitors of parasites that are resistant to Complex III inhibitors that are in clinical use or development, indicating that they could be used in treating drug resistant parasites. In sum, we have developed a versatile, scalable approach to screen for compounds that target the ETC in apicomplexan parasites, and used this to identify and characterize novel inhibitors. Author summary: Apicomplexan parasites impart major health and economic burdens on human societies. Treatment options against these parasites, which include the causative agents of toxoplasmosis (Toxoplasma gondii) and malaria (Plasmodium spp.), are limited. The apicomplexan mitochondrial electron transport chain is critical for parasite proliferation and pathogenesis, and is a validated drug target. In this study, we develop a powerful suite of approaches for screening compound libraries to identify and characterize electron transport chain inhibitors in these parasites that are potent, chemically diverse, and active against drug resistant strains of the parasites. These approaches enable us to distinguish between on-target inhibitors and those that cause non-specific parasite death, and allow us to pin-point the molecular target of inhibitors. We employ these approaches to identify an inhibitor with dual molecular targets. The novel compounds we identify represent new pathways towards much needed treatments for the diseases caused by apicomplexans. [ABSTRACT FROM AUTHOR]