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Inflammation is one of the body’s defensive reactions against microorganisms and allergens. The NF-κB pathway is an essential component of the inflammatory responses involved in a number of respiratory tract diseases, such as asthma and chronic obstructive pulmonary disease. In traditional medicine, Eucalyptus globulus Labill. (Myrtaceae) is used in inhalation therapy for respiratory tract infections and inflammation. In this study, the anti-inflammatory activity of Eucalyptus globulus essential oil and its impact on the NF-κB pathway was evaluated. A rat model of carrageenan-induced hind paw edema was used to evaluate the anti-inflammatory activity of E. globulus oil. Essential oil was injected intraperitoneally at doses of 0.5-3 mg/kg. An in vitro model based on a luciferase reporter gene construct (with repeats of a consensus NF-κB coding sequence) was used for investigation of the inhibitory effects of oil on the NF-κB pathway at concentrations of 100-250 μg/ml in Human chronic myeloid leukemia (K562) cells. E. globulus oil significantly reduced rat hind paw edema (P<0.05) at doses of 0.5 and 1.0 mg/kg at 1 to 3 h after carrageenan injection. The maximal reduction in edema was 58%. E. globules oil inhibited TNFα-induced NF-κB activity significantly (P<0.01) at a concentration of 250 μg/ml. The findings of the present study show that E. globulus essentialoil is a potent anti-inflammatory natural product, whose effects could be mediated through the NF-κB pathway.