The primary pathway of the detoxification of aflatoxin (AF) metabolites occurring at the end of phase-1 biotransformation is glutathione (GSH) conjugation via glutathione-S-transferase (GST) enzyme in phase-2. In this study, the activity of Nigella sativa seeds (NS) and thymoquinone (TQ) on phase-2 detoxification pathways of AF was investigated in light of GSH, GST alpha-3 (GSTA3), and AFB1-DNA adducts detected by the immunohistochemical method in broilers’ livers. One-hundred twenty chickens at one-day-old were divided into six groups as the control (CNT), AF, TQ, NS, AF + TQ, and AF + NS groups and fed for 28 days. AF, TQ, and NS were added to the relevant diets at 2 mg/kg, 300 mg/kg, and 5%, respectively. The administration of AF alone strongly stimulated the formation of AFB1-DNA adduct and reduced GSH and GSTA3 levels in hepatocytes (p < 0,01). In contrast, TQ and NS were found to reduce significantly the amount of AFB1-DNA adducts in AF + TQ and AF + NS groups (p < 0,01). We concluded that NS and TQ achieved this by increasing GSH and GSTA3 levels (p < 0,01) thanks to their antioxidant properties, and hence detoxifying the reactive metabolites of AF. Also, we consider that the AFB1-DNA adduct constituted in 28 days can be used as a biomarker for exposure to AF in broilers.