The antibacterial activity of 35 isolates of Lentinula edodes, a shiitake mushroom, against Bacillus subtilis was evaluated by diffusion technique in agar with a semi-solid overlay. All isolates inhibited B. subtilis and the isolate Le1 promoted the formation of the largest inhibition zone. L. edodes Le1 also presented antibacterial activity against foodborne pathogens and food contaminant bacteria, particularly Gram-positive species. The antibacterial activity of the culture filtrate after 18-25 days of cultivation of L. edodes in broth at 25degreesC was high. The inhibitory activity was observed only in the organic layer when the culture filtrate was partitioned between ethyl acetate and water, suggesting that the inhibitory substances have low polarity. The silica gel thin-layer zone at Rf values of 0.63-0.80, developed in chloroform - acetone ethyl acetate - methanol 40:5:5:2, was responsible for the antibacterial activity against B. subtilis. The inhibitory activity of L. edodes was detectable in the culture filtrate after heat treatment at 100degreesC for 10 min and after storage at 4degreesC for 120 days