This study investigated the in vitro and in vivo anti-hepatocellular carcinoma activity of pseurotin A isolated from the n-butanol extract of Aspergillus fumigatus Fr. AUMC 8002 (nBE-AF). The in vitro anticancer activity of nBE-AF was measured against a human hepatocellular carcinoma cell line (HepG2) using a sulforhodamine-B (SRB) assay. The intraperitoneal median lethal dose (LD50) of nBE-AF was determined in rats. Hepatocellular carcinoma was induced in rats by a single intraperitoneal injection of diethylnitrosamine (DEN)(200 mg/kg b. wt.) followed by subcutaneous injections of carbon tetrachloride (CCl4)(3 ml/kgb. wt.) weekly for 6 weeks. After administration of these carcinogens, 1/10 and 1/20 LD50 doses of nBE-AF were administered intraperitoneally daily. NBE-AF exhibited significant cytotoxic activity against HepG2 cells. Administration of DEN and CCl4 significantly elevated the serum levels of liver function and tumour markers and significantly downregulated tumour necrosis factor-α gene expression. Moreover, DEN and CCl4 decreased immunohistochemical Bax expression and increased Bcl-2 expression in the liver. Co-treatment with nBE-AF mitigated the DEN+ CCl4-induced alterations in a dose-dependent manner. Histopathological evaluation of the liver substantiated the above biochemical results. These results confirmed that nBE-AF, via its major isolated secondary metabolite, pseurotin A, exerted an anti-hepatocarcinogenic effect and could be used as a chemopreventive agent for hepatocellular carcinoma. awareness of the role of mycobacteria in infections associated with exposure to aquarium fish.