The primary goal of the present research investigation is the development and evaluation of thymoquinone-loaded nanoemulsion (TMQ-NE) for glaucoma therapy. The Box-Behnken Design approach was used for optimization of the TMQ-NE formulation. The TMQ-NE was prepared with the low-energy method, and was evaluated for its physicochemical properties revealed 29.10 ± 0.87 nm droplet size with 0.096 ± 0.03 of polydispersity index. The TMQ-NE exhibited the 6.2 ± 0.2 pH and 302 ± 1.5mOsm/kg osmolarity. Release studies demonstrated that TMQ-NE released 84.87 ± 0.18% of thymoquinone, while ex vivo corneal permeability study showed around 90% of permeation. The safety and efficacy assessments were conducted using ex vivo HET-CAM assay and in vivo Draize test, where the formulation was found safe for ocular administration. The optimized TMQ-NE exhibited a significant reduction in intraocular pressure (IOP) by 39.59 ± 0.88% within 1.5 h post-administration in animal model. Conclusively, the findings promote thymoquinone as an optimistic treatment option for glaucoma, underscoring the potential of TMQ-NE as an effective ocular drug delivery system.