The objective of this study was to investigate chemical composition, antimicrobial and cytotoxic activities of dried methanol and dichloromethane extracts of the flowering tops (herb) of yarrow, Achillea millefolium L. (Asteraceae) collected from natural habitat in Serbia. This plant, predominantly distributed in the northern hemisphere, is widely used in folk medicine, but also as an important crop for pharmaceutical and cosmetic industries, primarily due to its appetite-stimulating, choleretic, gastroprotective, spasmolytic, analgesic, antiinflammatory, astringent, antioxidant and/or antimicrobial activities. GC-FID and GC-MS analysis of unsaponifiable fraction of dichloromethane extract showed that the most abundant compounds were alpha-amyrin, (1-sitosterol, one taraxasterol isomer and (1-amyrin. LC-MS analysis of methanol extract revealed 28 phenolic compounds, predominantly caffeoylquinic acids, as well as apigenin and luteolin type flavonoids. Further, microdilution assay showed that both extracts possess moderate antimicrobial activity against Listeria monocytogenes (both MICs 1.25 mg mL-1) and Candida albicans (MICs 2.5 and 0.3125 mg mL-1, respectively). Investigation of the antibiofilm effects showed that methanol extract significantly inhibited biofilm formation of L. monocytogenes, but did not disrupt L. monocytogenes mature biofilm. Additionally, the reduction in L. monocytogenes motility was induced by methanol extract in dose dependent manner. Furthermore, MTT assay showed that cancer A549 and HCT 116 cell lines were more sensitive to the action of dichloromethane extract (both IC50 values 0.7 mg mL-1). Both extracts exhibited greater toxicity to cancer than to normal MRC-5 cells, as well as selective antimicrobial effect against L. monocytogenes and C. albicans. The results of bioactivity testing of chemically characterized A. millefolium extracts represent good basis for further investigations of this crop with the aim of widening its industrial application and justifying cultivation of the investigated yarrow population.