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Occupational exposure to the waste anaesthetic gases (WAGs) is a crucial problem for healthcare personnel. Cancer is among the potential long-term adverse effects of WAGs. The present occupational molecular epidemiology study was conducted in healthcare personnel (anaesthetists, nurses and technicians; n = 46), working in operating rooms (ORs; n = 34) and recovery units (RUs; n = 12) of the same hospital, to assess the genotoxicity risk of WAGs exposure. Twenty-one healthy available hospital staff allocated to other wards, without the history of working in ORs and RUs were the control group. A micronucleus test was carried out for peripheral blood lymphocytes (PBLs) and buccal epithelial cells (BECs). Exposure to the anaesthetics was assessed with sevoflurane concentrations and inorganic fluoride levels in post-shift urine samples of the healthcare staff. As an exposure marker, sevoflurane concentrations in ORs and RUs were measured using passive samplers. The micronuclei frequencies were increased in both PBLs (approximately two times) and BECs (approximately three times) of the healthcare personnel. Urinary sevoflurane concentrations exceeded the biological equivalent level in 23 personnel. Air sevoflurane levels in the breathing zone in three ORs and one RU did not exceed the established occupational exposure limits. Both in surrogate tissue (PBLs) and in target tissue (BECs) of the personnel of RUs and ORs of the same hospital, the genotoxicity risk was evident and similar. Originality of this study, in addition to the WAGs exposure confirmation of the healthcare personnel, was the involvement of the RU personnel for the genotoxicity assessment, which was the first time in the scientific literature.