Mushrooms are one of the few foods that are high in β-glucan. β-glucan's cholesterol lowering properties from oat and barley is well established, but it's not clear whether mushroom β-glucan has similar functionality. We aimed to analyse the β-glucan content in Australian mushrooms and evaluate their bile acid binding capacity, the primary cholesterol lowering mechanism of β-glucan. Raw, boiled and fried Australian grown Agaricus bisporus (button, cup, flat and brown mushrooms) and Pleurotus spp. (shimeji and oyster) were freeze dried and the β-glucan content determined using a Megazyme kit. Oat β-glucan was measured using an Association of Official Agricultural Chemists method (AOAC 995.16). An in vitro digestion method was used to assess bile acid binding capacity of mushroom and oat β-glucans. ^(1,2) Samples were analysed in triplicate and statistically compared using ANOVA. The β-glucan content of freeze-dried raw A. bisporus mushrooms (4.5 to 8.1 g/100g) were similar to that of oats (7.6 g/100g, all p > 0.05), whereas Pleurotus mushrooms contained ~5x more β-glucan (32.5–37.4 g, p < 0.05). As mushrooms are high in moisture (~90%), β-glucan content was much lower in fresh A. bisporus mushrooms compared to oats (0.3–0.7% v. 6.9%, respectively, p < 0.05) while Pleurotus mushrooms contained 3.2% (oyster) and 3.7% (shimeji) β-glucan. Boiling increased β-glucan content of oyster, button, flat and brown mushrooms by 3–7% (p < 0.05) but frying had no effect and neither cooking method affected β-glucan content of shimeji or cup mushrooms. Bile acid binding capacity of A. bisporus mushrooms (29–36%) was equivalent to raw oats (36%, p > 0.05) whereas the bile acid binding capacity of oyster mushrooms (22%) was lower than oats (p < 0.05). Cooking increased bile acid binding capacity which was related to changes in β-glucan content. Serving sizes of 150–300 g raw, 80–200 g cooked or 10–20 g freeze dried A. bisporus mushrooms were estimated to provide 1 g of β-glucan (amount required per serving of oats or barley for a high-level health claim related to cholesterol lowering) compared to 30 g raw, 20–40 g cooked or 3 g freeze-dried Pleurotus mushrooms and 14 g raw oats. In conclusion, although Pleurotus mushrooms had higher β-glucan levels compared to A. bisporus and oats, their bile acid binding capacity was significantly lower. A. bisporus (button, cup, flat, brown) mushrooms had similar bile acid binding properties to oats, but levels of β-glucan in fresh mushrooms were low. The cholesterol lowering effects of mushrooms, and acceptability of consumption at required levels, needs to be confirmed by clinical trials.