Bacterial blotch, caused by Pseudomonas tolaassiand related species, may be influenced by the microbial community of the mushroom cap. The objectives of this work were to compare methods for bacterial assemblage extraction and to assess the bacterial microbiome in mushroom caps with and without blotch symptoms. Mushrooms with blotch symptoms (n=50) and asymptomatic mushrooms (n=50) were collected at an organic farm in Pennsylvania and processed using sonication (resulting in washate and post-washate samples) or a whole mushroom processing strategy, prior to DNA extractions. The V4 hypervariable region of the 16S rRNA gene was sequenced using Illumina MiSeq. The washate and whole mushroom methods showed differences in the microbiome depending on the mushroom health status. However, regardless of treatments, Proteobacteria and Bacteroidetes were the most predominant phyla. An overrepresentation in the relative abundance for the classes Gammaproteobacteria and Aphaproteobacteria was found for the washate and post-washate methods, respectively. Forty-nine exact sequence variants (ESV) were related to Pseudomonasspp., from which thirteen, three, and one ESV grouped, respectively, with blotch pathogens, P. tolaasii, P. agariciand P. constantinii. Bacterial populations differed depending on the isolation method and the mushroom health status.