The isolation of Ewingella americana, an unusual Enterobacteriaceae, is reported here for the first time in a non-animal reservoir. Thirty-five strains of E. americana have been recovered from the cultivated mushroom, Agaricus bisporus. The biochemical characteristics of these strains are consistent with previously published descriptions of this species recovered from clinical specimens and from molluscs. DNA reassociation analysis was used to confirm the identity of mushroom-derived E. americana, and restriction fragment length polymorphism was used to reliably differentiate strains that otherwise demonstrated little phenotypic variation.