The ethanolic extracts obtained from the macrofungus Xylaria polymorpha (Pers.) Grev. (Ascomycetes) have been investigated for their antimicrobial activity. Growth inhibition using agar disc diffusion assays was determined against Escherichia coli ATCC 11230, S. aureus ATCC 6538P, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 8427, Bacillus cereus ATCC 7064, Micrococcus luteus CCM 169, Candida albicans ATCC 10231, Rhodotorula rubra DSM 70403, Geotrichum capitatum ATCC 28576, Debaryomyces hansenii DSM 70238, Kluyveromyces fragilis ATCC 8608 and Cryptococcus neoformans ATCC 90112. As a result of study, we have found that X. polymorpha revealed antimicrobial activity against all tested microorganisms. Especially, G. capitatum is more susceptible to the extracts.