The aim of present study was to establish standardization parameters for pharmacognostic evaluation of Macrocybe crassa, a wild edible mushroom of West Bengal. For this purpose, microscopic features of powder such as physical characters, spore measurement, type of hyphae were examined and recorded. Physico-chemical parameters like organoleptic features, loss on drying (10.53%), total ash (17.04%), swelling capacity (66.66 ml/g), water holding capacity (174.69 g/g), water solubility index (406.65%) and fluorescent behaviour against 16 reagents were also determined. Alcoholic extract was prepared using methanol as solvent where extractive value was 27.52%. Preliminary mycochemical analysis indicated the presence of cardiac glycoside, carbohydrate, flavonoid, phenol, saponin and terpenoids while absence of alkaloid and steroid in methanol extract. While quantitatively major bioactive components were present in the following order of flavonoid> phenol> ascorbic acid> β-carotene> lycopene. In addition, HPTLC and HPLC profiles were recorded to determine phenolic fingerprint. HPTLC characterization showed presence of at least 5 components as detected by scanning the plate at 300 nm. HPLC chromatogram of the extract indicated existence of minimum 14 peaks which might be of phenolic compounds. Along with, DPPH radical scavenging activity (EC50 value 2.455 mg/ml) and total antioxidant capacity (7.4 ± 1.24 μg AAE/mg of extract) were determined to evaluate antioxidant activity. The above parameters, which are being reported for the first time, are significant towards establishing the pharmacognostic standards for future identification and authentication of genuine mushroom material. © 2015, RJPT All right reserved.