Antibacterial activity of Lentinula edodes against Bacillus subtilis was evaluated in cell-free filtrates obtained after growth in 14 different culture media. The highest B. subtilis growth inhibition was promoted by filtrates of growth media supplemented with rice bran, vermiculite or molasses. L. edodes dry mycelial biomass in liquid culture with 0.5% added rice bran was 3.2 mg/ml, after growth for 30 days at 25ºC without shaking, and 4.3 mg/ml under orbital shaking (150 rpm). However, antibacterial activity, detected between 20 and 24 days of incubation of stationary cultures, was absent in filtrates of aerated cultures. Temperatures of 20-25ºC enhanced both growth and antibacterial activity. Optimum pH for L. edodes mycelial growth was 3.0-3.5, while for production of antibacterial substance(s) it was 4.5. Our results indicated that incubation conditions that enhance mycelial growth are quite different from those necessary for production of antibacterial substance(s) by L. edodes