The purpose of this study was to determine the role of heat shock protein 72 (Hsp72) changes in cardiac injury caused by microwave radiation, aimed at providing novel insights into the mechanism of this damage. A digital thermometer was used to measure the rectal temperature of the rats' pre- and post-radiation. On the 1st, 7th, 14th, and 28th days post-radiation, the changes in electrocardiogram (ECG) were analyzed by a multi-channel physiological recorder. The myocardial enzyme activities and ion concentrations were detected by an automatic biochemical analyzer. Additionally, the levels of myocardial injury markers were established by the enzyme-linked immunosorbent assay (ELISA), and those of hormones were measured by radioimmunoassay. The structure and ultrastructure of the myocardial tissue were observed using an optical microscope and transmission electron microscopy (TEM). The expression of Hsp72 was measured by Western blot and immunofluorescence analyses. Post-exposure, the rectal temperature in the R-group increased significantly, ECG was disordered, and the concentrations of ions were decreased. Furthermore, the activities of myocardial enzymes were changed, and the contents of myocardial injury markers and hormones were increased. We observed damage to the structure and ultrastructure and significantly increased expression of Hsp72. As a whole, the results indicated that S-wave microwave radiation at 30 mW/cm2 for 35 min resulted in damage to the cardiac functionality organigram, caused by a combination of the thermal and nonthermal effects.