Chemotherapy is the primary means of treating protozoan parasitic infections. A problem for chemotherapy is to find a novel and potential molecule in protozoa, which could be exploited as drug target. To reach this goal, mitochondrion of protozoa can be considered as the most valuable and potential organelle because of its unique structure and function compared to their natural host habitat. In fact, the respiratory systems of parasitic protozoa typically show greater diversity in electron pathways than do their host animals. These unique aspects of electron transport chain (ETC) complexes and their related enzymes represent promising targets for chemotherapy. A cytochrome independent Alternative Oxidase (AOX) in parasites is a leading drug target. Topoisomerases play key functions in replication and organization of kDNA, which is present in a specialized region of unique mitochondria known as kinetoplast. They are considered as potential targets for anti-parasitic drugs. Moreover, a novel pathway of type II Fatty acid synthesis in mitochondria of trypanosomatids provides a new array of inhibitors that could be effective against these parasites. Recent studies on the emergence of drug resistance severely limit the arsenal of available drugs against protozoan parasites. Particularly, mutations of cytochrome b gene of ETC or changes in iron homeostasis by mitochondrial enzyme aconitase alter sensitivity of MDR1 and regulate resistance level to anti-parasitic drugs. This review summarizes recent state of our knowledge and understanding of the action of various therapeutically applied substances on mitochondria and their potential application in the future.