The antioxidant activity and the anti-α-amylase and anti-acetylcholinesterase capacities of secondary metabolites from different organs (roots, stems, leaves and flowers) of Tunisian Satureja barceloi were determined. The variation in the distribution of phenolic metabolites among roots, stems, leaves and flowers extracts of S. barceloi with various solvent systems (methanol, ethyl acetate, hexane and distilled water) has not been characterized before. Significant variation of phenolic compounds was observed according to organs rather than to extracting solvents. The analyzed organs show a high level of phenolic compounds although the stems contains the highest total polyphenols (132.53±0.48 mg AGE/g Ex), flavonoids (48.99±0.65 mg RE/g Ex) and flavonols (34.93±0.29 mg QE/g Ex) contents. The phenolic fraction was dominated by sagerinic acid, caffeic acid glucoside and epigallocatechin, detected using HPLC-PDA/ESI-MS. The antioxidant activity of all extracts, evaluated by four in vitro tests, was high and varied significantly according to the type of solvent used and the plant organ. The aqueous extracts of leaves exhibited the greatest inhibitory effect on alpha-amylase while the methanolic extract of leaves and stems revealed the most important acetylcholinesterase inhibitory effect. Hence, S. barceloi extracts could be used as a source of various bioactive molecules in pharmaceutical industry.