The parameters to extract polysaccharide-enriched fractions (PEF) from mushrooms using MAE (microwave-assisted extraction) were adjusted following a full factorial 32 experimental design. The highest yield and total carbohydrate values, using Lentinula edodes as model mushroom, were obtained at 180 °C and 30 min. Several mushroom species were submitted to MAE and their PEF yields ranged between 12.1-44.2%. (1→3)-β-Glucans determination using a conventional fluorimetric method changed depending on the standard utilized. NMR analyses of PEF indicated that the presence of other polysaccharides in the extracts or their specific folding, might impair the proper determination of (1→3) linkages by the fluorophore. Mushrooms from Cantharellales order contained (1→3)-β-glucans but they were not detected with the fluorimetric method. Therefore, although the method (after adjustments) was sensitive enough to detect their presence in many mushroom extracts, it cannot be used for all species and it is also not recommended for quantitative determinations.