In traditional medicine, the genus Achillea is utilized for its anti-inflammatory, antispasmodic, and diuretic properties. Achillea aucheri Boiss., species native to Iranian flora was studied due to the Achillea genus's rich supply of phytochemical substances and the absence of essential phytochemical investigations.The methanolic extract (ME) from aerial parts of A. aucheri was fractionated using a C-18 pre-packed cartridge and vacuum liquid chromatography, followed by chromatographic separation using a reversed-phase preparative HPLC. The isolated components were structurally elucidated using 1D and 2D-NMR spectroscopy. Furthermore, essential oil from A. aucheri was isolated through hydrodistillation. Analysis of the essential oil was done through gas chromatography–mass spectrometry (GC-MS) and GC-FID. Free-radical scavenging activity, total flavonoid and phenolic content of the ME were determined using the DPPH assay, AlCl3, and Folin-Ciocalteu reagents, respectively. Anti-microbial activity of the A. aucheri essential oil was investigated against different bacteria and fungus via disk diffusion and micro-dilution methods.Structure elucidation of the purified compounds revealed presence of one major flavonoid (1) and a sesquiterpene (2) in ME. From the A. aucheri essential oil, α-thujone (45.6%), artemisia alcohol (26.5%), and yomogi alcohol (8.8%) were the major constituents. The total flavonoid, phenolic content of the ME were 39.70 ± 0.05 mgQE/gE and 66.70 ± 0.08 mgGAE/gE, respectively, while IC50 value in DPPH assay was 86.28 ± 0.01 μg/mL. Concerning the achieved antimicrobial activity, maximum diameter of inhibition zone of the essential oil was obtained against S. aureus (31.5 ± 0.5 mm) followed by S. epidermidis (13.5 ± 0.7 mm) and M. luteus (11.5 ± 0.5 mm). Additionally, the minimum inhibitory concentration (MIC) values for S. aureus, S. epidermidis, and M. luteus were recorded as 2.5, 2.5 and 1.25% (v/v), respectively. Exhibited activity could be explained to the presence of α-thujone, artemisia alcohol and yomogi alcohol, the most abundant phytochemicals of A. aucheri essential oil.