Our previous study demonstrated that hyperbaric oxygen (HBO) improves heart function predominantly through reducing oxygen stress, modulating energy metabolism and inhibiting cell apoptosis. The present study aimed to investigate the protective effects of HBO on mitochondrial function and autophagy using rats with a ligated left anterior descending artery. The cardioprotective effects of HBO were mainly evaluated using ELISA, fluorescent probes, transmission electron microscopy and reverse transcription‑quantitative PCR (RT‑qPCR). HBO pretreatment for 14 days (once a day) using a 0.25 MPa chamber improved mitochondrial morphology and decreased the number of autophagic vesicles, as observed using a transmission electron microscope. HBO pretreatment significantly increased the levels of ATP, ADP, energy charge and the opening of the mitochondrial permeability transition pore, but decreased the levels of AMP, cytochrome c and reactive oxygen species. Moreover, HBO pretreatment significantly increased the gene or protein expression levels of eIF4E‑binding protein 1, mammalian target of rapamycin (mTOR), mitochondrial DNA, NADH dehydrogenase subunit 1, mitofusin 1 and mitofusin 2, whereas it decreased the gene or protein expression levels of autophagy‑related 5 (Atg5), cytochrome c, dynamin‑related protein 1 and p53, as determined using RT‑qPCR or immunohistochemistry. In conclusion, HBO treatment was observed to protect cardiomyocytes during myocardial ischemia‑reperfusion injury (MIRI) by preventing mitochondrial dysfunction and inhibiting autophagy. Thus, these results provide novel evidence to support the use of HBO as a potential agent for the mitigation of MIRI.