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This study aimed to investigate the seasonal variation in the chemical composition and antimicrobial activity of Achillea fragrantissima essential oil (EO). EO was collected at 10 days intervals, from May to July 2014, for a total of seven harvests. The EO was extracted from aerial parts by hydro-distillation for 3 hours. The antimicrobial activity was evaluated by well diffusion method against three bacterial species (Escherichia coli, Staphylococcus aureus and Bacillus subtilis and two fungal species (Candida albicans and Aspergillus niger). The chemical composition of EO was performed by gas chromatography-mass spectrometry (GC-MS). The activity of the essential oils against the tested bacteria and fungi varied depending on harvesting time of A. fragrantissima. In general, the highest activity of A. fragrantissima essential oils was against Gram-positive bacteria at T6 (the end of June). The chemical constituents of EO showed quantitative and qualitative variation of several specific chemical compounds, the ratio of identified compounds range between 92.29 and 95.88%, the major fluctuations in the composition of EO also revealed the presence of trans-sabinyl acetate (0.75-10.20%), -terpineol (3.53-9.39%), trans-Menth-2-en-1-ol (6.5-13.34%), and -thujone (11.34-22.11). In this study, the antimicrobial activity and the chemical composition of the EO extracted from A. fragrantissima were affected significantly by the time of collection. T6 period would be the best time to harvest A. fragrantissima plants to obtain EO with the highest antimicrobial activity. The chemical difference noted among seasons may explain the different in antimicrobial activity.