Mitochondrial DNA (mtDNA) mutations contribute to various diseases characterized by low ATP production. In contrast, thyroid hormone (T₃) induces mitochondrial biogenesis and enhances ATP generation. To evaluate the role of T₃-mediated mitochondrial biogenesis in patients with mtDNA mutations, three fibroblast cell lines with mtDNA mutations were evaluated, including two patients with Leighs Syndrome (LS) and one with Hypertrophic Cardiomyopathy (HCM). Compared to control cells, patient fibroblasts displayed similar levels of mitochondrial mass, PGC-1, Tfam and UCP2 protein expression. Patient cells exhibited a 1.6-fold elevation in reactive oxygen species (ROS) production, a 1.7-fold elevation in cytoplasmic Ca²⁺ levels, a 1.2-fold elevation in mitochondrial membrane potential and 30% less complex V activity compared to controls. Patient cells also displayed 20-25% reductions in both cytochrome c oxidase (COX) activity and MnSOD protein levels compared to controls. Following T₃ treatment of patient cells, ROS production was decreased by 40%, cytoplasmic Ca²⁺ was reduced by 20%, COX activity was increased by 1.3-fold, and ATP levels were elevated by 1.6-fold, despite the absence of a change in mitochondrial mass. There were no significant alterations in the protein expression of PGC-1, Tfam or UCP2 in either T₃-treated patient or control cells. However, T₃ restored the mitochondrial membrane potential, complex V activity and the levels of MnSOD to normal values in patient cells. These results suggest that T₃ acts to reduce cellular oxidative stress, which may help attenuate ROS-mediated damage, along with improving mitochondrial function and energy status in cells with mtDNA defects.