The mitochondria within human cells play a major role in a variety of critical processes involved in cell survival and death. An understanding of mitochondrial involvement in various human diseases has generated an appreciable amount of interest in exploring this organelle as a potential drug target. As a result, a number of strategies to probe and combat mitochondria-associated diseases have emerged. Access to mitochondria-specific delivery vectors has allowed the study of biological processes within this intracellular compartment with a heightened level of specificity. In this review, we summarize the features of existing delivery vectors developed for targeting probes and therapeutics to this highly impermeable organelle. We also discuss the major applications of mitochondrial targeting of bioactive molecules, which include the detection and treatment of oxidative damage, combating bacterial infections, and the development of new therapeutic approaches for cancer. Future directions include the assessment of the therapeutic benefit achieved by mitochondrial targeting for treatment of disease in vivo. In addition, the availability of mitochondria-specific chemical probes will allow the elucidation of the details of biological processes that occur within this cellular compartment.