This study aims to investigate the antioxidant and anti-inflammatory activities of Thymus quinquecostatus Celak essential oil (TQEO) and explore its underlying mechanisms. The antioxidant capacity was evaluated through DPPH, hydroxyl, and superoxide anion radicals scavenging assays. Flow cytometry was employed to analyze the effects of TQEO on apoptosis and cell cycle progression in oxidative stress-induced cells, while ELISA was employed to assess its regulatory effects on key inflammatory cytokines. Chemical composition analysis was performed using gas chromatography-mass spectrometry (GC-MS). The results demonstrated that TQEO exhibited significant scavenging activity on DPPH, hydroxyl, and superoxide anion radicals and effectively reduced intracellular reactive oxygen species  levels in oxidatively damaged cells. Anti-inflammatory evaluation revealed that TQEO dose-dependent inhibited of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6. GC-MS analysis identified its major bioactive constituents, including thymol, carvacrol, terpinen-4-ol, eugenol, terpineol, linalool and menthol. These findings confirm the remarkable antioxidant and anti-inflammatory properties of TQEO and characterize its chemical composition, providing a theoretical foundation for understanding its pharmacodynamic basis and mechanisms.