The aim of this study was to evaluate for the first time total phenolic content, antioxidant, antimicrobial and cytotoxic effects of heptane, chloroform and methanol extracts from Achillea multifida (DC.) Boiss. (Compositae) aerial parts followed by identification of phenolic compounds in the methanol extract, which showed the highest total amount of phenolics, antioxidant and antibacterial activity. The phenolic compounds in methanol extract were identified for the first time by ESI-Q-TOF LC/MS. The phenolic compounds were identified as: chlorogenic acid; quercetin hexoside; luteolin-7-O glucoside; dicaffeoyl quinic acid; luteolin. The chloroform extract showed strong activity and selectivity against HeLa, HT-29 and MCF-7 cell lines. The results indicated that low concentrations (100 mu g/mL and 50 mu g/mL) of chloroform and methanol extracts had no toxic effects on the NIH/3T3 cells. Therefore, the methanol extract from A. multifida aerial parts may be a natural source of antioxidants and antibacterial and seems to have potential application in both medicine and the food industry. In additon, the chloroform extract might be a potential anti-cancer agent.