Human oral exposure to aflatoxin B (AFB ) and fumonisin B (FB ) is associated with increased hepatocellular carcinoma. Although evidence suggested interactive AFB -FB hepatotoxicity, the underlying mechanisms remain mostly unidentified. This work was aimed at evaluating the possible AFB -FB interplay to induce genetic and cell cycle toxicities in BRL-3A rat hepatocytes, reactive oxygen species (ROS) involvement, and the AFB metabolizing pathways cytochrome P450 (CYP) and arachidonic acid (ArAc) metabolism as ROS contributors. Flow cytometry of stained BRL-3A hepatocytes was used to study the cell cycle (propidium iodide), ROS intracellular production (DCFH-DA, HE, DAF-2 DA), and phospholipase A activity (staining with bis-BODIPY FL C11-PC). The CYP1A activity was assessed by the 7-ethoxyresorufin-O-deethylase (EROD) assay. Despite a 48-h exposure to FB (30 μM) not being genotoxic, the AFB (20 μM)-induced micronucleus frequency was overcome by the AFB -FB mixture (MIX), presumably showing toxin interaction. The mycotoxins blocked G1/S-phase, but only MIX caused cell death. Overall, the oxidative stress led these alterations as the pretreatment with N-acetyl-l-cysteine reduced such toxic effects. While AFB had a major input to the MIX pro-oxidant activity, with CYP and ArAc metabolism being ROS contributors, these pathways were not involved in the FB -elicited weak oxidative stress. The MIX-induced micronucleus frequency in N-acetyl-l-cysteine pretreated cells was greater than that caused by AFB without antioxidants, suggesting enhanced AFB direct genotoxicity probably owing to the higher CYP activity and ArAc metabolism found in MIX. The metabolic pathways modulation by AFB -FB mixtures could raise its hepatocarcinogenic properties.