The axenic culture of Thymus decussatus, a rare wild species found only above 1500 m altitude in the St. Katherine Protectorate, Egypt, was established. Full MS (Murashige and Skoog medium) medium supplemented with 0.35 mg/l benzyladenine (BA) and 0.2 mg/l kinetin (Kin) was found to be optimal for micropropagation and acclimatization. Hexane extract showed the most significant cytotoxic effects against hepatocellular carcinoma HepG2 cells with an IC50 of 12 ± 0.69 μg/ml and selectivity over normal hepatocytes (THLE2 IC50=38.57 ± 1.65 μg/ml). High-performance liquid chromatography identified the main components as flavonoids and phenolic acids, with rosmarinic acid being the most abundant (23.7 μg/g dry weight). The combination of hexane extract with sorafenib had a synergistic effect, decreasing the IC50 from 9.289 ± 0.51 to 2.274 ± 0.13 μg/ml. In contrast, an antagonistic effect was seen with doxorubicin, increasing its IC50 from 0.716 ± 0.04 μg/ml to 6.499 ± 0.36 μg/ml. The hexane extract exhibited S-phase cell cycle arrest and apoptosis induction. Analysis of gene expression profiles showed upregulation of p53 and MDR1 genes. Molecular docking analysis revealed that the flavonoids quercetin, chlorogenic acid, apigenin, and rosmarinic acid have high-affinity binding to pro- and anti-apoptotic proteins, suggesting possible mechanisms of action. In conclusion, this work supports conserving this rare species and investigating its phytochemicals as potential adjuvants to improve hepatocellular carcinoma treatment.