Aging is associated with decreased mitochondrial content and function in skeletal muscle, possibly due to compromised biogenesis and autophagic removal of dysfunctional mitochondria. The aim of this study was to compare markers of mitochondrial content and biogenesis and of autophagy between skeletal muscle from young and aged American Quarter Horses. Citrate synthase protein and mtDNA copy number were decreased in triceps brachii (TB) muscle (P < 0.05) from aged horses, suggesting an age-related decline in mitochondrial content. Concomitantly, mRNA expression of PGC-1α and TFAM, regulators of mitochondrial biogenesis, was lower in aged compared to young TB (P < 0.05). Expression of autophagy markers suggested an age-associated decline of autophagy. The autophagosomal cargo protein SQSTM/p62 accumulated with age in both muscles (P < 0.05). Expression of Autophagy-related protein Atg5 (P < 0.05) and the autophagosome-bound form of Microtubule-associated protein light chain 3 (LC3-II; P < 0.05) were lower in aged compared to young TB. While LC3 transcript level was elevated in aged compared to young GM (P < 0.001), protein expression of LC3-II was unaffected. Gene expression of Lysosomal Membrane-Associated Protein 2 (LAMP2) was not affected by age in either muscle. However, LAMP2 protein expression declined with age (P < 0.05), suggesting a decline in autophagosome-lysosome fusion. Taken together, our data indicate that equine skeletal muscle mitochondrial content and biogenesis were impaired with age. Further, autophagosome formation and lysosomal degradation were negatively affected in aged TB and GM, respectively. Future research needs to explore whether interventions targeting these cellular processes can prolong health and performance of aging American Quarter Horses.