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α-Amanitin, the main toxic substance from mushroom species (Amanita genus), blocks the activity of RNA polymerase II (Pol II) in mammalian cells causing inhibition of transcription and subsequent synthesis of structural and enzymatic proteins. It has been postulated that α-amanitin generates the increase of reactive oxygen species (ROS) concentration. The micronucleus (MN) test was used on an animal experimental model to evaluate possible potential genotoxic effect of α-amanitin on mice bone marrow cells. At the same time the activity of antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT) as well as concentration of thiobarbituric acid reactive substance (TBARS) were investigated in the lysate of mice erythrocytes. α-Amanitin was administered intraperitoneally at the doses: 0.1, 0.15, and 0.25 mg/kg bw (LD50 for mice) 48 h prior to sacrification. A statistically significant increase of SOD activity was observed in the hemolysate for all the investigated α-amanitin doses as compared to the negative control (p < 0.05). CAT activity for α-amanitin doses 0.1 and 0.15 mg/kg was higher in comparison to the negative control but the differences were not statistically significant (p > 0.05). However, for the dose 0.25 mg/kg the activity of CAT was statistically significantly higher (p < 0.001). All the tested α-amanitin doses decreased TBARS concentration in the hemolysate as compared to the negative control but the differences were not statistically significant (p > 0.05). A statistically significant increase of mean values of MN percent was found in polychromatic erythrocytes (PCEs) as compared to the negative control for α-amanitin dose 0.1 and 0.25 mg/kg (p < 0.05). For the dose 0.15 mg/kg the mean value of MN percent was higher but it did not demonstrate statistical significance (p > 0.1). The observed disturbances in the activity of the examined antioxidant enzymes in cells exposed in vivo to α-amanitin suggest indirect genotoxic effect of α-amanitin through ROS generation.α-Amanitin disturbed the activity of the SOD and CAT in mice erythrocytes. Frequency of micronuclei exposured to α-amanitin in two tested doses was increased. We suggest indirect genotoxic effect of α-amanitin through ROS generation.