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This study aimed to identify the bioactive compounds of the ethyl acetate extract of Aspergillus niger SH2-EGY using GC-MS and to evaluate their protective role against aflatoxin B 1 (AFB 1 )-induced oxidative stress, genotoxicity and cytotoxicity in rats. Six groups of male Sprague-Dawley rats were treated orally for 4 weeks included the control group, AFB 1 -treated group (80 μg/kg b.w); fungal extract (FE)-treated groups at low (140) or high dose (280) mg/kg b.w and the groups treated with AFB 1 plus FE at the two tested doses. The GC-MS analysis identified 26 compounds. The major compounds found were 1,2,3,4,6-Penta-trimethylsilyl Glucopyranose, Fmoc-L-3-(2-Naphthyl)-alanine, D-(-)-Fructopyranose, pentakis (trimethylsilyl) ether, bis (2-ethylhexyl) phthalate, trimethylsilyl ether-glucitol, and octadecanamide, N-(2- methylpropyl)-N-nitroso. The in vivo results showed that AFB 1 significantly increased serum ALT, AST, creatinine, uric acid, urea, cholesterol, triglycerides, LDL, carcinoembryonic antigen, alpha-fetoprotein, interleukin-6, Malondialdehyde, nitric oxide, Bax, caspase-3 and P53 mRNA expression, chromosomal aberrations and DNA fragmentation. It decreased serum TP, albumin, HDL, Bcl-2 mRNA expression, hepatic and renal TAC, SOD and GPx content and induced histological changes in the liver and kidney. FE prevented these disturbances in a dosage-dependent manner. It could be concluded that A. niger SH2-EGY extract is safe a promising agent for pharmaceutical and food industries., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)