This work aimed to determine the chemical composition of hydrosol extract from aerial parts of Atractylis gummifera and to assess the in vitro antioxidant and anti-inflammatory properties of this extract, as well as the isolated carlina oxide from the hydrosol extract of Atractylis gummifera. The study also examined the in vitro synergistic effect of carlina oxide with BHT and diclofenac to reduce their minimum effective doses and minimize potential side effects. The hydrosol extract was analyzed using Gas Chromatography (GC) and Gas Chromatography Mass Spectroscopy (GC/MS). The antioxidant activity was evaluated using radical scavenging activity (DPPH) and the β-carotene bleaching test, with BHT as the positive control. The anti-inflammatory activity was assessed using the protein denaturation method, with diclofenac serving as the positive control. The primary component of the hydrosol extract from A. gummifera was identified as the acetylenic compound carlina oxide (79.1%), which was isolated and confirmed through spectroscopic techniques such as 1H NMR, 13C NMR, and IR. The results of the biological assays demonstrated that both the hydrosol extract and carlina oxide exhibited noteworthy antioxidant and anti-inflammatory properties. Additionally, combining carlina oxide with positive controls yielded enhanced activities, resulting in a significant reduction in inhibitory concentrations and doses of synthetic antioxidants and anti-inflammatories. Further research is warranted to explore the potential applications of these positive control combinations involving carlina oxide in the pharmaceutical industry