Anesthetic isoflurane, which is increasingly used in the medical and scientific fields, remains a poorly studied halogenated agent whose adverse effects are not fully understood. It appears to be related to stress and oxidative damage in biological tissues originating from reactive oxygen species (ROS) generated during metabolism. Considering its common use in research laboratories, whether for performing surgical procedures or for prior exposure to euthanasia, knowledge about its interference in liver biochemical parameters of exposed experimental models is important. This study aimed to evaluate the effect of acute exposure to isoflurane, for a short period prior to euthanasia, or after exposure to a sixty-minute period, on the concentrations of ROS and thiobarbituric acid reactive substances, and enzyme activity of catalase (CAT) and superoxide dismutase in mouse liver tissue. Isoflurane reduced CAT activity in the liver tissue of acutely exposed individuals and anesthetic interference was maintained for at least 20 days after anesthetic exposure. Our results demonstrate that acute anesthetic exposure can impair biochemical liver experiments aimed at analyzing antioxidant parameters.