A novel monoacylglycerol with an amino acid moiety, neomastoidin A (1), was isolated from the fruiting bodies of the poisonous mushroom Macrolepiota neomastoidea. The structure of 1 was established by extensive spectroscopic analysis and further confirmed by application of a dibenzoate chirality method. Neomastoidin A (1) exhibited cytotoxicity against SK-OV-3 and SK-MEL-2 cell lines.