Doxorubicin (Dox), an anthracycline antibiotic, is a drug that inhibits the replication of DNA and metabolic processes in cancer cells that have high proliferative potential, thereby acting as an anti-cancer agent. Although Dox can cause severe side effects such as myocardial damage and heart failure, the exact mechanism by which it leads to myocardial injury remains uncertain. In this study, we examined the impact of Dox on the mitochondrial quality control system and the regulation of mitochondrial respiration and autophagy in H9c2 rat myoblast cells cultured in vitro using western blotting, immunohistochemistry, the Seahorse XF24 system, and flow cytometry. Our research sheds light on the processes that drive the influence of Dox on mitochondrial function. We showed that Dox does not hinder the start of autophagic flux or the functioning of lysosomes. However, Dox affected the mitochondrial quality control system, causing a shift towards fission and impairing the regulation of mitochondrial respiration. This led to an increase in oxidative stress and the inhibition of autophagy, especially the fusion of autophagosomes with lysosomes. The inhibition led to a substantial decline in autolysosome formation, which was attributed to the buildup of dysfunctional mitochondria and a subsequent surge in oxidative stress, resulting in heightened myocardial cell death.