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In this study, nutritional value, free amino acids, phenolic and sugar compounds, mineral and heavy metal contents of truffles, Terfezia olbiensis, and Terfezia claveryi from Turkey were chemically characterized using different analytical methods. In vitro antioxidant characteristics, beta-carotene bleaching assay, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity, ABTS cation radical decolorization assay, and the anticholinesterase activities, against AChE and BChE enzymes, were evaluated. Individual phenolic ingredients and free amino acids were analyzed using UPLC-ESI-MS/MS. HPLC-RID was used to assess sugar profile, and ICP-MS was used to determine minerals and heavy metals. Gentisic acid (25.48 and 14.84 mu g/kg dry weight (dw)), protocatechuic acid (21.55 and 15.54 mu g/kg dw), and p-hydroxy benzoic acid (18.07 and 16.99 mu g/kg dw) were quantified in T. olbiensis and T. claveryi, respectively. Rhamnose, fructose, trehalose, and mannitol were the sugars identified. However, T. olbiensis and T. claveryi revealed high amounts of essential amino acids (310.25 and 314.08 mg/100 g, respectively). In addition, the ethyl acetate extracts of the mushrooms performed highest radical scavenging activity, whereas the hexane extracts of mushrooms displayed highest lipid peroxidation inhibitory activity. The results showed that the tested extracts obtained from Terfezia species can be conveniently used as sources for the protection of lipid peroxidation, free radicals, and moderate anticholinesterase agents in pharmaceutic and as food additives for extension of products’ shelf life food and dietary supplement industries. Furthermore, the techniques used in this study were easy, safe, and sensitive.