Lentinus squarrosulus (Mont.) is a wide edible mushroom widely consumed in Thailand. Ethanol extract was extracted from mycelium of L. squarrosulus No. 2 and Lentinus basidiocarp obtained from the local market at Mahasarakham province using 50% ethanol solvent. The effect of ethanolic extracts was tested for inhibition to some pathogenic bacteria. Antibacterial activity was tested by Poison food technique, it was found that 50 % ethanol extract from mycelium at concentrations of 10%, 15%, and 30% was effective to inhibit Bacillus cereus at the highest level (+++) and mycelium extract at the concentrations of 15% and 30% can inhibit Staphylococcus aureus with low level (+). But all concentrations of L. squarrosulus basidiocarp extract unable to inhibit the growth of B. cereus, Escherichia coli, Pseudomonas aeruginosa and S. aureus. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of ethanolic extract from mycelium inhibit B. cereus was 125 mg/ml. The results indicate that the potential of L. squarrosulus mycelium is an excellent source of antimicrobial agents.