Medical herbs have many bioactive component and they are used in microbial treatment since ancient times. The resistance of pathogens to antibiotics became a critical problem, so researches for novel antimicrobial agents are required. Anacyclus pyrethrum (pellitory, Spanish chamomile or Mount Atlas daisy) is commonly used as a traditional medicine, therefore the antimicrobial activity of the root of this medicinal plant was investigated against 17 bacteria and 1 fungi by using disk diffusion method. The test strains include Bacillus, Enterobacter, Enterococcus, Escherichia, Klebsiella, Listeria, Pseudomonas, Salmonella, Staphylococcus and Candida genera. Besides, chemical composition of this sample was determined by Gas Chromatography-Mass Spectroscopy analysis. The results were presented that A. pyrethrum has antimicrobial activity against all tested microbial species except E. faecalis and S. typhimurium. Eighteen major chemical components were determined, but some composition of this sample is not match with library. For this reason, this medicinal plant contain unknown molecules and this molecules should be analysed NMR spectra for 3D structure determination and identification.