The antibacterial activities of thirty mushroom species belonging to Basidiomycetes and Ascomycetes, cultivated on two liquid media, were evaluated against gram-positive (Bacillus subtilis and Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria by the disk diffusion method. All of the mushrooms, except Auriporia aurea, Fomes fomentarius, and Lyophyllum shimeji, showed different antibacterial activity levels-from 9.5 mm in diameter of the inhibition zone to full inhibition of growth of the test bacteria. The antibacterial activities of Crinipellis schevczenkovi, Hohenbuehelia myxotricha, Oxyporus obducens, and Spongipellis litschaueri were observed for the first time. The antibacterial potential of culture liquids of the investigated species was higher than that of their mycelia activity. Dependence of the intensity of antibacterial activity on the culture medium was shown. The antibacterial efficiency of the most active species (Lentinus edodes, Piptoporus betulinus, and Phellinus igniarius) was verified and compared with those of some commercial antibiotics and natural essential oils of Salvia and Eucalyptus. The culture liquid of Piptoporus betulinus, obtained after cultivation on glucose-peptone-yeast culture medium, is a potential substance for further creation of antibacterial products.