Background: Doxorubicin (DOX) is generally recognized to have important cardiotoxic side effects. Studies are contradictory about the interaction between hyperbaric oxygen (HBO2) therapy and doxorubicin-induced cardiomyotoxicity. Recent data suggests that HBO2 therapy can lead to preconditioning of myocardium while generating oxidative stress. Herein we have investigated the effect of HBO2 therapy in a DOX-induced cardiomyocyte injury animal model. Methods: Twenty-one rats were divided into three equal groups as follows: 1) Group 1 is a control group (without any intervention), used for evaluating the basal cardiac structures and determining the normal value of cardiacs and serum oxidative markers; 2) Group 2 is the doxorubicin group (single dose i.p. 20 mg/kg doxorubicin) for detecting the cardiotoxic and systemic effects of doxorubicin; 3) Group 3 is the doxorubicin and HBO2 group (100% oxygen at 2.5 atmospheric for 90 minutes, daily), for evaluating the effect of HBO2 in doxorubicin induced cardiotoxicity. At the end of the protocols, the hearts were harvested and blood samples (2 ml) were obtained. Results: The doxorubicin treated animals (Group 2) had increased oxidative stress markers (both cardiac and serum) and severe cardiac injury as compared to the basal findings in the control group. Nevertheless, the highest cardiac oxidative stress index was detected in Group 3 (control vs. Group 3, p = 0.01). However, histological examination revealed that cardiac structures were well preserved in Group 3 when compared with Group 2. Conclusions: Our results suggest that HBO2 preconditioning appears to be protective in the doxorubicin-induced cardiotoxicity model. Future studies are required to better elucidate the basis of this preconditioning effect of HBO2.