In this paper the antimicrobial activity of 1 year old crude ethanolic extracts obtained from Cordyceps sinesis, Laricifomes officinalis, Oudemansiella mucida and Coprinus comatus were investigated. The antimicrobial activities of extracts against two Gram-positive bacteria (Bacillus thuringiensis, Staphylococcus aureus) and two Gram-negative bacteria (Klebsiella pneumoniae, Enterobacter aerogenes) were determined by disk diffusion and microbroth dilution method according by EUCAST in 96-well microplates. Microorganisms were obtained from Czech Collection of Microorganisms. Absorbance after and before the experiment were substracted, converted to binary system and obtained values to Probit analysis were used. Not all macromycetes ethanolic extracts showed antimicrobial activity against tested bacteria. Antimicrobial activity determined by MIC methodology showed extracts from Oudemansiella mucida, Cordyceps sinesis, Coprinus comatus in the tested range. Conversely, the best antimicrobial activity tested by disc diffusion methods showed extract from Laricifomes officinalis. Equally, more better studying of antimicrobial activity in these mushrooms will needed.