This research study was conducted to investigate the cardioprotective activity of alcoholic extract of Ganoderma lucidum (GL) against cyclophosphamide (CYP)-induced cardiotoxicity in rats model. Therewithal, the regulation in the expression level of Aquaporin (AQP) water channels in the heart was evaluated. Cardiotoxicity was induced in Wistar rats by administering a single-dose injection of CYP (200 mg/kg, i.p.) on the seventh day of the experimental period. GL (500 mg/kg, gavage) was administered daily for seven days. Cardiac alteration following CYP and GL administration was evaluated using electrocardiographic changes, morphological staining, AQP gene expression and western blot analysis. It was observed that the CYP administration significantly (p < 0.001) increased cardiac troponin- I (cTn-I) and elevated the level of creatine kinase isoenzyme MB (CK-MB). Further, rats treated with CYP showed increased expression levels of AQP-3, -4 and - 7 compared to the control group. The treatment with GL significantly (p < 0.001) reversed the level of cardiac biomarkers in CYP-induced cardiotoxicity. Potential cardioprotective effect of GL which reduced the severity of cellular damage of the myocardium was supported by histopathological examination. The biochemical, expressional and histopathological reports supported the cardioprotective activity of GL which could be attributed to changes in myocardial edema. To the best knowledge of the authors, this is the first study that reports the cardioprotective role of GL in CYP-induced myocardial edema and the changes in the expression levels of AQP.