A gram-negative bacterium was isolated from spent substrates of Agaricus bisporus and showed significant antagonistic activity against Pseudomonas agarici. The bacterium was identified as Alcaligenes sp. based on cultural, biochemical, physiological characteristics and a 16S rRNA sequence analysis. The isolate is saprophytic, but not parasitic or pathogenic on cultivated mushroom, whereas it showed strong inhibitory effects against P. agarici cells in vitro. The control efficacy of Alcaligenes sp. HC12 against brown blotch of P. agarici was up to 63% on Agaricus bisporus. The suppressive bacterium may be useful for the development of biocontrol systems.