A key characteristic of mushroom polysaccharides that elicit an immunomodulatory response is that they are rich in β-glucans and low in α-glucans. In this study we analysed nine commercially available preparations from three mushroom species, Reishi (Ganoderma lucidum), Shiitake (Lentinula edodes) and Maitake (Grifola frondosa), for β- and α-glucan content. Based on β- and α-glucan content we selected three extracts to combine into a formula and evaluated the ability of the individual extracts and formula to impact on the expression of cytokines IL-1α, IL-6, IL-10 and TNF-α in human macrophages with and without LPS stimulation. The majority of mushroom extracts and the formula were found to be highly potent immuno-stimulators possessing EC50 values lower than 100 μg/mL. Interestingly the mushroom formula had lower EC50 values in TNF-α expression from LPS stimulated macrophages compared to the individual extracts, suggesting a potential synergistic effect of the mushroom formula. A response additivity graph and curve-shift analysis illustrated that indeed the mushroom formula exhibited an immuno-stimulatory synergistic effect on the expression of the majority of cytokines evaluated in both LPS stimulated and non-stimulated human macrophages, with IL-10 having an antagonistic response. This study represents the first report of a synergistic immuno-modulatory response in human macrophages elicited from a mushroom formula rationally derived from β- and α-glucan content.