Medicinal mushrooms belonging to Lentinus spp. exhibit significant antibacterial activities, but little attention has been paid to their efficacy against the food-borne pathogen, Bacillus cereus. The present study for the first time quantitatively evaluated the antibacterial activity of different extracts from fruiting bodies of a well-authenticated Iranian native strain of medicinal mushroom, Lentinus tigrinus, against Gram-positive spore-forming bacterium B. cereus. The findings revealed that the acetone extract inhibited the growth of B. cereus at concentrations as low as 31.25 μg/ML, while it had no effect against Escherichia coli and Staphylococcus aureus even at 10,000 μg/ML. The rest of the bacteria were also susceptible to the acetone extract at concentrations greater than 5 mg/ML. Antibacterial activities of the methanol-ethyl acetate extract and the hot water extract were significantly weaker than that of the acetone extract, which contained high amounts of total phenols (5.83 ± 0.08 mg GAE/g, dw), while Fourier transform infrared spectroscopy (FT-IR) confirmed the presence of functional groups, such as hydroxyl, carbonyl, amide, and amine. Further studies by scanning electron microscopy (SEM) confirmed obvious changes in the morphology of B. cereus in response to the acetone extract of L. tigrinus. This study may suggest that L. tigrinus could be a good natural source for isolating and purifying antibacterial compounds against B. cereus.