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Background: The genus Thymus is one of the well-known members of the Lamiaceae, comprising about 215 species. It is a polymorphic genus in this family. Therefore, possible differences in chemical composition among polymorphs may result in differences in their biological activities. Objective: This study was designed to specify chemical composition in order to assess antioxidant activities and to characterize active constituents of essential oil of Thymus pubescens. Methods: Essential oil was collected by hydrodistillation and was analyzed using GC-MS and GCFID. Antioxidant activities were evaluated by ABTS radical cation scavenging and β-carotene bleaching inhibition methods. Detection and identification of antioxidant components were performed by TLC-bioautography assay using ABTS•+ reagent. Results: GC analyses indicated the presence of 39 components, accounting for 96.64% of total constituents, with thymol (38.67%), γ-terpinene (7.46%), and p-cymene (5.54%) as the main components. The IC50 values for antioxidant activities were equal to 1.15 (1.05 – 1.25) μg/mL and 0.45 (0.33 – 0.62) μg/mL for ABTS•+ scavenging and β-carotene bleaching inhibition tests, respectively. TLC-bioautography of oil resulted in the identification of thymol as the major antioxidant compound. Conclusion: Essential oil exhibited very potent and significant antioxidant activities correlated well with increasing dose in vitro.