Antioxidants have been considered essential for preventing cell damage by scavenging deleterious free radicals. The consumption of antioxidant-rich plants is associated with a reduced risk of some chronic diseases. This study evaluates the antioxidant and acetylcholinesterase inhibition activities of aqueous extracts obtained from different parts of Diplotaxis simplex and Diplotaxis harra from Tunisia. The study also aimed to investigate the action of simulated gastrointestinal juice on antioxidant activities of both extracts. The total phenolic, flavone and flavonol, and flavanone and dihydroflavonol contents were determined by Folin–Ciocalteau, aluminum chloride and 2,4-dinitrophenylhydrazine colorimetric methods, respectively. The metal ion chelating activity, acetylcholinesterase inhibition capacity, and free radical scavenging potential of the extracts towards ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl), hydroxyl, superoxide and nitric oxide were also evaluated. The action of simulated gastro-intestinal fluids on the flavone and flavonol content and total antioxidant activity of the flower extracts was surveyed. Extracts from the seeds and flowers of D. simplex and D. harra displayed the highest amounts of phenols (2691.7 and 2694.5 mg Caffeic Acid Equivalent (CAE)/100 mg; 3433.4 and 2647.2 mg CAE/100 mg, respectively) and flavonols/flavones (2144.4 and 2061.1 mg Rutin Equivalent (RE)/100 g; 1922.6 and 1461.1 mg RE/100 g, respectively). The flower and seed extracts exhibited the highest rates of antioxidant and acetylcholinesterase inhibition activities. A decrease in the flavonoid content and antioxidant activity was observed after extract exposure to simulated saliva. Antioxidant and acetylcholinesterase inhibition activities were noted to depend on plant species and plant parts. In vitro gastrointestinal digestion is useful in assessing the bio-accessibility of compounds with biological activities from food. The simulated gastrointestinal fluids influenced the flavonoid concentration and antioxidant activity.