Acute hepatitis is characterized by acute hepatic parenchymal inflammation, raising liver function indices, and offering little chance of successful treatment. Consequently, there is a lot of curiosity in the discovery of natural products as valuable alternatives to traditional anti-inflammatory agents with inhibitory effect, especially on cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LO). Black cumin seed oil (BCSO) possesses antioxidant and anti-inflammatory properties due to the constituent of thymoquinone (TQ). The protective anti-inflammatory properties of BCSO and its nanoemulsion formulation were investigated in mice that had been given lipopolysaccharide (LPS) to cause liver injury by hepatic inflammatory markers assessment on both gene and protein levels, molecular docking, and histological examination. Results showed that BCSO is a promising potent COX-2 inhibitor. The nanoemulsion was shown to enhance the anti-inflammatory effect of BCSO compared to Indomethacin as standard anti-inflammatory drug. In order to reduce these side effects, combinatorial therapy of BCSO with Indomethacin at comparatively lower doses of Indomethacin showed a potentiated anti-inflammatory effect. TQ showed enhanced binding affinity interaction to (5-LO) that may explain its potential inhibitory activity to COX-2. The current presented data suggests the potential anti-inflammatory effect of both BCSO and its nanoemulsion for wide scale clinical applications.