The extracts of aerial parts of Achillea clavennae, Achillea holosericea, Achillea lingulata and Achillea millefolium (hexane:ether:methanol=1:1:1) have been tested for antimicrobial activity in a disk diffusion assay against five bacteria (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Salmonella enteritidis) and two fungi (Aspergillus niger and Candida albicans). Extracts of all four species possessed a broad spectrum of antimicrobial activity against all tested strains. The composition of the extract of Achillea clavennae, which showed the strongest activity, was investigated and the structures of isolated compounds were elucidated by spectral means (1D and 2D NMR, UV, IR and MS). The extract yielded alkanes, fatty acids, monoterpenes, guaiane sesquiterpenes (rupicolin A and B, 1-deoxy-1alpha-peroxy-rupicolin A and B), and flavonoids (apigenin and centaureidin). This study confirms the ethnopharmacological use of plants from this genus and places Achillea clavennae L. on the same level as the recognized Achillea millefolium.