•We firstly reported mycelial sluggish growth disease of Flammulina filiformis.•Pseudomonas migulae is the pathogenic bacterium of this disease.•Molecular mechanism of F. filiformis in response to P. migulae was elucidated.•Hydrogen peroxide silver disinfectant can effectively inhibit P. migulae. Flammulina filiformis, an industrially cultivated edible mushroom worldwide, suffers serious losses in yield and quality owing to bacterial diseases. Here, we reported the mycelial sluggish growth disease (MSGD), a new bacteriosis on F. filiformis. Pseudomonas migulae was identified as the pathogen by 16S rRNA gene phylogenetic analysis and morphologic observation. Hydrogen peroxide silver disinfectant (HPSD) was shown to effectively inhibit P. migulae and alleviate the disease symptoms at 10.00 g/L concentration. Furthermore, the molecular mechanism of F. filiformis in response to P. migulae infection was elucidated by transcriptome analysis. In the early infection stage, the infection signals are transducted into the mycelia, thus activating a series of stress defense responses, including peroxidase activity, antibiotic synthesis, and drug catabolism. In the middle and late infection stages, the apoptosis signal is transducted through the mitochondrial pathway, causing damage to DNA in the cell nucleus, resulting in DNA repair, gene silencing, and negative regulation of macromolecule synthesis. Finally, proteins and polysaccharides are strongly hydrolyzed to accelerate cell senescence, death, and mycelial growth retardation. These findings provide references for understanding the interaction mechanism between edible mushroom and pathogenic bacterium and for controlling mushroom bacterial diseases.