Myocardial ischaemia-reperfusion (IR) injury poses a severe threat to cardiac health, particularly in the ageing population, where susceptibility to such damage is significantly heightened owing to age-related declines in mitochondrial function, thus highlighting mitochondria as crucial targets for innovative therapies. The aim of this study was to investigate the combined modality therapy involving mitochondrial transplantation and the mitochondrial boosters mitoquinone and melatonin to address myocardial IR injury in aged rats. A total of 54 male Wistar rats, aged 22-24 months, were randomly divided into groups that either received IR injury or not, and were subjected to various treatments, both individually and in combination. Myocardial IR injury was induced by temporarily blocking and reopening the left anterior descending coronary artery. Mitoquinone was given intraperitoneally for 14 days prior to ischaemia, while melatonin and isolated mitochondria were administered intraperitoneally and intramyocardially, respectively, at the onset of reperfusion. Finally, we evaluated changes in haemodynamic indices, creatine kinase-MB levels, mitochondrial function endpoints and the expression of mitochondrial biogenesis genes, including sirtuin 1 (SIRT-1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and nuclear respiratory factor 2 (NRF-2). The triple therapy enhanced myocardial function, decreased creatine kinase-MB levels and improved mitochondrial function along with the expression of mitochondrial biogenesis genes in aged IR rats. This combined approach elicited significant cardioprotection in comparison to single or dual therapies. The triple therapy provided substantial cardioprotection in aged rat hearts by improving mitochondrial function and biogenesis through enhanced SIRT-1/PGC-1α/NRF-2 profiles, suggesting a promising strategy for mitigating IR injury in elderly patients.