Medicinal mushrooms belonging to <i>Lentinus</i> spp. exhibit significant antibacterial activities, but little attention has been paid to their efficacy against the food-borne pathogen, <i>Bacillus cereus</i>. 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, <i>Lentinus tigrinus</i>, against Gram-positive spore-forming bacterium <i>B. cereus</i>. The findings revealed that the acetone extract inhibited the growth of <i>B. cereus</i> at concentrations as low as 31.25 μg/ML, while it had no effect against <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> 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 <i>B. cereus</i> in response to the acetone extract of <i>L. tigrinus</i>. This study may suggest that <i>L. tigrinus</i> could be a good natural source for isolating and purifying antibacterial compounds against <i>B. cereus</i>.
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