Ewingella americana was identified as the causal agent of internal stipe necrosis on symptomatic samples collected from mushroom farms located in Castilla-La Mancha, Spain, during the years 2008 to 2010. Six isolates obtained from Agaricus bisporus (button mushroom) and Pleurotus ostreatus (oyster mushroom) were studied to gain insight into their diversity. According to their biochemical profile, the isolates corresponded to biogroup 1. RAPD typing was performed directly with broth cultures of bacteria using two selected primers, and the profiles obtained were used to define four RAPD types. The pathogenicity of isolates was tested by artificial inoculation in A. bisporus and P. ostreatus, in both of which the pathogen reproduced the disease symptoms, and was reisolated from inoculated specimens. Artificial inoculations of the bacteria on Pleurotus eryngii (king oyster mushroom) reproduced the symptoms of the disease, but, until now, this bacterium has not been isolated from this fungus.