Background: 5-Fluorouracil (5-FU) is one of the commonly used anti-cancer drugs. However, it ranks as the second most common drug that causes cardiotoxicity. Ganoderma lucidum (G.L.) is a mushroom used for centuries for its different therapeutic properties. The aim of the study is to investigate the potential cardioprotective effect of G.L. against 5-FU cardiotoxicity, anti-inflammatory, and antioxidant properties. Material and methods: Thirty male Albino rats were divided into five groups. The control group was given normal saline orally for 14 days. The second group was treated as the control for 13 days and then 100 mg/kg 5-FU was administered intraperitoneally on day 14. The third group received G.L. 100 mg/kg orally for 13 days followed by a single 100 mg/kg 5-FU intraperitoneally on day 14. The fourth group was treated with 2 mg/kg enalapril orally for 13 days followed by a single 100 mg/kg 5-FU intraperitoneally on day 14. The last group received G.L. 100 mg/kg orally for 14 days. On day 15 the animals were euthanized, and blood was collected for biochemical analysis of cardiac biomarkers (troponin (TNNI3) and heart-type fatty acid binding protein (H-FABP)), oxidative stress markers (total antioxidant capacity (TAC) and malondialdehyde (MDA)), and the pro-inflammatory marker (tumor necrosis factor-alpha (TNF-alpha)). The heart tissue was isolated for the histopathological investigation of cyclooxygenase-2 (COX-2) expression. Results: 5-FU administration has led to an increase in the level of H-FABP, TNNI3, MDA, TNF-alpha, and COX-2 expression while it has significantly reduced the level of TAC. G.L. could prevent the 5-FU-induced cardiotoxicity via its effect on all the measured parameters. Conclusion: G.L. can potentially offers cardioprotection against 5-FU-induced cardiotoxicity through its antioxidant and anti-inflammatory effects.