The cholesterol lowering properties of oats and barley, attributed to their high beta-glucan content, are well established, but it remains unclear whether mushrooms, also rich in beta-glucan, exhibit a similar functionality. We aimed to quantify the beta-glucan content of commonly consumed Australian mushrooms and evaluated their bile acid binding capacity, the primary cholesterol lowering mechanism of beta-glucan. Raw, boiled and fried Australian grown Agaricus bisporus (button, cup, flat and brown mushrooms) and Pleurotus spp. (shimeji and oyster) along with oats were freeze-dried and the beta-glucan content was determined. The bile acid binding capacity of these samples was assessed using an in vitro digestion assay. The beta-glucan content of freeze-dried raw A. bisporus mushrooms (4.5-8.1 g per 100 g) was similar to that of oats (7.6 g per 100 g, all p > 0.05), whereas Pleurotus mushrooms contained similar to 5 times more beta-glucan (32.5-37.4 g, p < 0.05). Boiling increased the beta-glucan content of oyster, button, flat and brown mushrooms by 3-7% (p < 0.05), but did not affect the beta-glucan content of shimeji or cup mushrooms. Frying had no effect on any mushroom type. The bile acid binding capacity of A. bisporus mushrooms (29-36%) was equivalent to that of raw oats (36%, p > 0.05), whereas the bile acid binding capacity of oyster mushrooms (22%) was lower than that of oats (p < 0.05). Both boiling and frying increased the bile acid binding capacity. The cholesterol lowering effects of A. bisporus mushrooms and the acceptability of consumption at the required levels need to be confirmed by clinical trials.