Abstract Previous missions to the lunar surface implicated potential dangers of lunar soil. In future explorations, astronauts may spend weeks or months on the Moon, increasing the risk of inhaling lunar dust. In an effort to understand the biological impact of lunar regolith, cell cultures derived from lung or neuronal cells were challenged with lunar soil simulants to assess cell survival and genotoxicity. Lunar soil simulants were capable of causing cell death and DNA damage in neuronal and lung cell lines, and freshly crushed lunar soil simulants were more effective at causing cell death and DNA damage than were simulants as received from the supplier. The ability of the simulants to generate reactive oxygen species in aqueous suspensions was not correlated with their cytotoxic or genotoxic affects. Furthermore, the cytotoxicity was not correlated with the accumulation of detectable DNA lesions. These results determine that lunar soil simulants are, with variable activity, cytotoxic and genotoxic to both neuronal and lung‐derived cells in culture.