In recent decades, toxic cyanobacterial blooms have become frequent in the drinking water supply and have caused serious deleterious effects to domestic and wild animals, as well as to humans. Two strains of the cyanobacterium species Cylindrospermopsis raciborskii (T2 and T3) were isolated from the Billings Reservoir (São Paulo, Brazil) and cultured in the laboratory for use in acute toxicity tests with mice, micro crustaceans, and fish. The results showed high toxicity of both strains in mouse bioassays (median lethal dose [LD50]; 24 h = 9.6 and 27 mg/kg; intraperitoneal injections). The symptomatology presented by mice was typical of neurotoxicosis, such as trembling, ataxia, convulsions and death by respiratory arrest. Acute and chronic effects were observed in Daphnia similis and Ceriodaphnia dubia, such as immobilization and reduced fitness, respectively. Although acute effects were not detected on the adult fish Danio rerio, chronic toxicity was observed for its larval stage. Although both strains showed high toxicity to all organisms, no consistent pattern was seen between the different bioassays and strains. The results also showed that C. raciborskii toxins are stable to heat and to extreme pH variations. Because of high toxicity of these strains and the potential risk to human health, the authors propose a revision of the legislation regarding safety factors for drinking water supply.