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Morchella fluvialis, a morel mushroom, is one of the most famous edible mushrooms all over the world. Interest in this mushroom is steadily increasing due to its organoleptic properties and nutritional value. The methanolic extract of M. fluvialis showed α-glucosidase inhibitory and antioxidant activities in an assay system. Therefore, the purification and characterisation of bioactive metabolites and evaluation of biological activity were conducted. Fractionation of the M. fluvialis extract resulted in the isolation of nine compounds, namely, three fatty acids, (9Z,12Z)-octadecadienoic acid (linoleic acid, 1), (9Z,12Z)-3-hydroxyoctadecadienoic acid (2) and (6Z,9Z)-13-hydroxyoctadecadienoic acid (3); four sterols, stellasterol (4), ergosterol peroxide (5), ergosterol (6) and brassicasterol (7); one sugar alcohol, arabitol (8); and nicotinamide (9). Among them, compounds 2–3 and 7 were first reported from Morchella. In addition, compound 1 exhibited potent α-glucosidase inhibition, with an IC50 value of 14.8 μM. The content of compound 1, the major compound, was 1.2 mg · g–1 extract, as quantitated by HPLC analysis, which was lower than the IC50 value of compound 1. Therefore, M. fluvialis can benefit from diabetes and related diseases through the synergistic effect of linoleic acid (1) and other ingredients.