Alpha-amanitin is a known cytotoxic substance found in some mushroom species including Amanita phalloides. Its main mechanism of action is to block the transcription, which can lead to cell death. Lack of reports on the genotoxicity of this toxin was an inspiration for undertaking this experiment. Genotoxic effect of α-amanitin on balb/c mice bone marrow cells was tested using: comet assay and chromosomal aberration test. The tested substance was given once by intraperitoneal administration to animals at doses: 0.1 mg/kg, 0.15 mg/kg and 0.25 mg/kg (LD50) body weight with 48 h exposure. The comet assay demonstrated a statistically significant increase in DNA damage for all the investigated α-amanitin doses compared to the negative control (p < 0.0001). The exposure to 0.15 and 0.25 mg/kg doses of α-amanitin also generated a statistically significant increase in the frequency of chromosomal aberrations in bone marrow cells of mice compared to the negative control (p < 0.05). The genotoxic effect induced by α-amanitin in mammalian cells can result in genome instability and its functional consequences.