Abstract Objectives The aim of this study was to investigate the effects of occupational exposure to anesthetic gases on peripheral mononuclear leukocyte DNA damage and oxidative status in operating room personnel. Design and methods The population of study includes 30 operating room personnel (Group I) and 30 non-operating room personnel for control groups (Group II). DNA damage was assessed by alkaline comet assay in peripheral lymphocyte. Plasma levels of total antioxidant status (TAS), total oxidative status (TOS) were determined by using a novel automated measurement method and oxidative stress index (OSI) was calculated (OSI was calculated as: OSI = (TOS / TAS) × 100). Results Mononuclear leukocyte DNA damage, plasma TOS and OSI were significantly higher in Group I than Group II [ p < 0.01, p < 0.05, p < 0.05 respectively]. There was also significantly decrease in plasma TAS in Group I than Group II [ p < 0.05]. There were positive significant correlations between DNA damage and TOS and OSI in Group I [ r = 0.84, p < 0.001, r = 0.89, p < 0.001 respectively]. Conclusions Our findings demonstrate that, mononuclear leukocyte DNA damage was significantly higher in operating room personnel. The increase of DNA damage might depend on oxidative effects of anesthetics.