This research was conducted to investigate the anti-biofilm and antimicrobial activities of essential oil from Achillea millefolium L. (EOAM) on clinical isolates of Staphylococcus aureus, as well as its chemical composition, and cytotoxic activity. The chemical composition of EOAM was analyzed by gas chromatography. Antimicrobial and antibiofilm activities were determined by broth micro-dilution method. Colorimetric assay was used to assess its cytotoxicity on human osteosarcoma (MG63), human breast cancer (MCF-7), and mouse fibroblast (L929) cell lines. Viability in the biofilm was studied using 2,3-bis (2- methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. 3-Cyclopenten-l-one, 2-hydroxy-3-(3-methyl-2-butenyl) (28.94 %), piperitone oxide (25.74 %), carvacrol (15.41 %), eucalyptol (5.16 %) and limonene (3.23 %) were the major components of the essential oil. ICso value was found to be 28.07 mu g/mL, 19.02 mu g/mL, 41.35 mu g/mL in the MCF-7, MG-63, and L929 cell line, respectively. Minimum inhibitory concentration (MIC) values were found in the range of 12.5-25 mu l/mL, whereas bactericidal activities reached higher concentrations values in the order of 25 and 50 mu l/mL. Minimal biofilm inhibition concentration (MBIC) value was found to be 1.56-12.5 (mu l/mL) while minimal biofilm eradication concentration (MBEC) value was found to be 6.25-50 (mu l/mL). EOAM damaged viability in the biofilm at MIC value by 35.3-94.3 %. These findings suggest that EOAM contains components that may be useful for the development of potential phytotherapeutic agents against S. aureus infections.