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Carvacrol (CAR), a naturally occurring phenolic monoterpene compound, has recently received attention for its potential use in food preservation. However, whether it is effective in controlling brown blotch disease caused by Pseudomonas tolaasii in edible mushrooms is unknown. The results of this study showed that CAR effectively inhibits and kills P. tolaasii in vitro by disrupting cell membrane integrity and causing the leakage of cellular components. Intracellular proteins and the DNA of P. tolaasii may not be the targets of CAR. CAR fumigation at a concentration as low as 20 μmol L−1 CAR effectively inhibited P. tolaasii-caused brown blotch disease in Agaricus bisporus, accompanied by a decrease in polyphenol oxidase activation, melanin production, and malondialdehyde accumulation. CAR treatment also significantly increased the activities of β-1,4-N-acetyl-glucosaminnidase, three antioxidant enzymes, and phenylpropanoid pathway-related enzymes, as well as promoting the accumulation of phenolic, flavonoid, and lignin substances in mushrooms, thereby inducing the resistance of mushrooms to the disease. These results demonstrate the potential application of carvacrol to control bacterial disease in A. bisporus mushrooms.