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Cyclophosphamide (CPA) is a widely used anti-neoplastic drug, but its usage is associated with several side effects including hepatotoxicity. This study was designed to evaluate the ameliorative effect of Ganoderma lucidium (GL) extract as a natural antioxidant against CPA hepatotoxicity in male Wistar albino rats. Four groups of rats (n = 7) were used in this study, Group I: served as a control group, group II: was administrated CPA (5 mg/kg), group III: received GL extract (50 mg/kg), and group IV: received CPA and GL extract at the same doses of groups II and III. All the treatments were orally administrated via gastric intubation for 21 consecutive days. Serum activities of the hepatic enzymes (AST, ALT and GGT) in addition to serum proteinogram were evaluated. Moreover, hepatic oxidant-antioxidant markers (4-hydroxynonenal (4-HNE), GSH, CAT and SOD) besides activity of cytochrome C oxidase enzyme were detected. Also, histopathological examination of the hepatic sections (H&E stained) was performed. Serum activity of liver enzymes was elevated, proteinogram was disturbed in CPA-treated animals which accompanied by an increase in hepatic content of 4-HNE, depletion of hepatic antioxidant (GSH, CAT, SOD) and inhibition of hepatic cytochrome C oxidase activity, also hepatic histopathological disruptions were noticed in this group. All these hepatic disturbances were partially ameliorated upon co-administration of GL extract with CPA. In conclusion, production of 4-HNE and inhibition of cytochrome C oxidase enzyme may play a role in occurrence of CPA hepatotoxicity and GL extract may have an ameliorative role against this toxicity.