Agaricus bisporus plays an important role in ecological processes and is one of the most widely cultivated mushrooms worldwide. Mushroom growth-promoting bacteria have been isolated from casing soil and compost, but microorganisms in the fruiting body have received only a little attention. To get an overview of phylogenetic diversity of microorganisms in the fruiting body of A. bisporus, as well as to screen antimicrobial and mushroom growth-promoting strains, and eventually intensify mushroom production, we isolated and characterized microorganisms from the fruiting body of A. bisporus. In total, 55 bacterial strains were isolated, among which nine isolates represented Actinomycetes. All the isolates were analyzed by 16S rRNA gene RFLP and sixteen representative strains by 16S rRNA gene sequencing. According to the phylogenetic analysis, eleven isolates represented the Gram-positive Bacillus, Lysinibacillus, Paenibacillus, Pandorea and Streptomyces genera, and five isolates belonged to the Gram-negative Alcaligenes and Pseudomonas genera. The bacteria isolated from the fruiting body of A. bisporus had broad-spectrum antimicrobial activities and potential mushroom growth-promoting abilities.