Thymoquinone (TQ), a bioactive compound found in Nigella sativa seeds, has gained considerable attention due to its potential therapeutic properties. This study aimed to isolate and characterize TQ from Nigella sativa essential oil and evaluate its antioxidant capacity, antibacterial activity, and cytotoxic effects on lung cancer A549 cells. Moreover, The binding potential of TQ to Keap1 (Kelch-like ECH associated protein 1) was investigated through molecular docking. The stability of the resulting complex was evaluated through molecular dynamics (MD) simulation. The CUPRAC assay revealed significant antioxidant capacity of TQ, as indicated by its high molar absorptivity coefficient. TQ also demonstrated notable free radical scavenging activity, with efficacy increasing with concentration. In terms of antiquorum sensing activity, TQ displayed effectiveness against all tested virulence factors in P. aeruginosa PAO1 and Chromobacterium violaceum in different range. Additionally, TQ induced cytotoxicity in non-small lung cancer cells, inhibiting cell viability. In conclusion, this study successfully isolated and characterized TQ from Nigella sativa seeds and demonstrated its remarkable antioxidant capacity, antibacterial activity, and cytotoxic effects on lung cancer cells. These findings suggest the potential of TQ for various applications in the food and pharmaceutical industries. The wet-lab study demonstrated that TQ had an antioxidant effect. The TQ was found to have high binding potential to Keap1 as it formed four conventional hydrogen bonds with the protein. The resulting Keap1-TQ complex was also found to be stable. Further research is warranted to explore the therapeutic potential of TQ and develop novel treatments based on its properties.