The metabolic and intracellular abnormalities in aging and diabetes cause loss of cardioprotection by routine interventions against myocardial ischemia/reperfusion (I/R) injury. We aimed to evaluate the possible interaction of aging and type-2 diabetes mellitus with cardioprotection and the potential protective effect of a mitochondrial cocktail (melatonin/nicotinamide mononucleotide (NMN)/ubiquinol) on myocardial I/R injury in aged diabetic rats.Male Wistar rats (n = 108, 22-24 months old, 400-450 g) received high-fat diet/low dose of streptozotocin to induce type-2 diabetes, then were randomized into 9 groups of 12 rats each with/without I/R and/or melatonin, NMN, and ubiquinol, alone or in dual or triple combinations. Myocardial I/R was induced by LAD occlusion for 30 min followed by 24 h reperfusion. NMN (100 mg/kg/48 h, intraperitoneally) was administered for 28 days before I/R operation. Melatonin (10 mg/kg, intraperitoneally) and/or ubiquinol (30 mg/kg, intravenously) were administered at early reperfusion. Finally, hemodynamic index changes, infarct size, CK-MB levels, mitochondrial functional endpoints, and expression of mitochondrial biogenesis genes (SIRT-1/PGC-1α/NRF-2/TFAM) were assessed.The solo and dual applications of melatonin, NMN, and ubiquinol did not exert remarkable cardioprotective impacts. However, the triple combination improved myocardial function and decreased infarct size and CK-MB levels following myocardial I/R (P < .05 to P < .01). It also improved mitochondrial function and restored mitochondrial biogenesis genes (P < .01).Combination therapy with melatonin, NMN, and ubiquinol exerted significant cardioprotection and improved mitochondrial function and biogenesis via upregulation of SIRT-1/PGC-1α/NRF-2/TFAM profiles in aged diabetic rats and, thus, offers a promising strategy for providing noticeable cardioprotection against I/R injury also in aged diabetic patients.