Hsp90 is one of the most conserved molecular chaperones ubiquitously expressed in normal cells and over-expressed in cancer cells. A pool of Hsp90 was found in cancer mitochondria and the expression of the mitochondrial Hsp90 homolog, TRAP1, was also elevated in many cancers. The mitochondrial pool of chaperones plays important roles in regulating mitochondrial integrity, protecting against oxidative stress, and inhibiting cell death. Pharmacological inactivation of the chaperones induced mitochondrial dysfunction and concomitant cell death selectively in cancer cells, suggesting they can be target proteins for the development of cancer therapeutics. Several drug candidates targeting TRAP1 and Hsp90 in the mitochondria have been developed and have shown strong cytotoxic activity in many cancers, but not in normal cells in vitro and in vivo. In this review, recent developments in the study of mitochondrial chaperones and the mitochondria-targeted chaperone inhibitors are discussed.