Antiretroviral therapy has dramatically reduced mortality in human immunodeficiency virus (HIV) infection. In 1988, the suggestion that the first antiretroviral drug, zidovudine, was the potential cause of muscle pathology in HIV-infected persons resulted in structural and biochemical patient studies demonstrating acquired mitochondrial dysfunction. Assessment of subsequent nucleoside analog reverse transcriptase inhibitor (NRTI) antiretroviral drugs has indicated that mitochondria are a common target of NRTI toxicity in multiple tissues, leading to a wide variety of pathology ranging from lipodystrophy to neuropathy. Overwhelmingly, these complications have emerged during post-licensing human studies. Subsequent animal and in vitro studies have then elucidated the potential pathological mechanisms, suggesting that NRTI-associated mitochondrial toxicity arises principally from inhibition of the sole mitochondrial DNA (mtDNA) polymerase gamma, leading to a reduction in mtDNA content (depletion). Millions of patients have been treated with mitochondrially toxic NRTIs and these drugs remain the backbone of antiretroviral rollout in much of sub-Saharan Africa. Here we describe the 25-year history of antiretroviral associated mitochondrial pathology and critically review the strength of evidence linking clinical, histopathological, and molecular data. We discuss recently described novel mechanisms of NRTI-associated mitochondrial damage and whether or not recently licensed NRTIs may be considered free from mitochondrial toxicity.