Objectives: To comparatively evaluate the effects of thymoquinone (TQ), the biologically active main component of volatile oil derived from Nigella sativa seeds, in an experimental dry eye model. Materials and Methods: A total of 36 BALB/c mice 10 weeks of age were used in the study. The mice were divided into 6 groups of 6 mice. Two groups were negative and positive controls, and the other 4 groups were treated with balanced salt solution, fluorometholone (FML), TQ, or vehicle (Tween80). After 1 week of treatment, the mice were killed and the eyes removed for histopathologic examination and cytokine analysis. Interleukin (IL)-1α tumor necrosis factor-α, interferon-γ, IL-2, IL-6, IL-10, and lactoferrin levels in the conjunctival tissue were measured by multiplex immunobead assay. The presence of inflammatory cells in ocular tissue samples were investigated by hematoxylin-eosin and periodic acid-Schiff staining. Inflammatory T cells containing CXT receptor in the conjunctiva were determined by flow cytometry. Results: FLML and TQ groups had less inflammatory cell density and more goblet cells compared to the other groups. High levels of IL-1α and IL-2 were found in the TQ group. Conclusion: TQ treatment was associated with reduced inflammation in pathological examination, but did not significant lower cytokine levels.