Recent studies have indicated that a major contributor to the innate immune enhancing properties of some medicinal plants is derived from the cell wall components of bacteria colonizing these plants. The purpose of the current study was to assess if the bacteria present within edible and medicinal mushrooms substantially contribute to the innate immune stimulating potential of these mushrooms. Whole mushrooms from thirteen types of edible fungi and individual parts from Agaricus bisporus were analyzed for in vitro macrophage activation as well as bacterial lipopolysaccharides (LPS) content, cell load, and community composition. Substantial variation between samples was observed in macrophage activation (over 500-fold), total bacterial load (over 200-fold), and LPS content (over 10 million-fold). Both LPS content (ρ = 0.832, p < 0.0001) and total bacterial load (ρ = 0.701, p < 0.0001) correlated significantly with macrophage activation in the whole mushroom extracts. Extract activity was negated by treatment with NaOH, conditions that inactivate LPS and other bacterial components. Significant correlations between macrophage activation and total bacterial load (ρ = 0.723, p = 0.0001) and LPS content (ρ = 0.951, p < 0.0001) were also observed between different tissues of Agaricus bisporus. Pseudomonas and Flavobacterium were the most prevalent genera identified in the different tissue parts and these taxa were significantly correlated with in vitro macrophage activation (ρ = 0.697, p < 0.0001 and ρ = 0.659, p = 0.0001, respectively). These results indicate that components derived from mushroom associated bacteria contribute substantially to the innate immune enhancing activity exhibited by mushrooms and may result in similar therapeutic actions as reported for ingestion of bacterial preparations such as probiotics.