A high-field NMR technique was used to analyze aqueous and organic extracts of truffles (Tuber aestivum vittadini) to characterize their chemical composition. Water-soluble metabolites belonging to different classes such as sugars, polyols, amino acids, and organic acids were almost completely assigned by means of one- and two-dimensional experiments (1H-1H COSY, TOCSY, 1H-13C HSQC, 1H-13C HMBC, and 1H-31P HMBC). The 1H spectral assignment of the cell membrane components such as lipids, sterols, and fatty acids extracted in organic solvents was also performed.