Wild samples were harvested from five different regions in Tunisia. Folin–Ciocalteu, aluminum chloride and vanillin methods were used to determine phenolic content. Individual phenolic compounds were identified using high-performance liquid chromatography. In vitro antioxidant capacity was investigated using three different tests (total antioxidant capacity, DPPH-radical scavenging activity and ABTS-radical scavenging activity). Total phenolic content varied from 1.31 mg GAE/g DR to 8.14 mg GAE/g DR; total flavonoid content ranged from 4.71 mg QE/g DR to 72.79 mg QE/g DR; and total tannin content was between 12.71 mg GAE/g DR and 105.61 mg GAE/g DR. HPLC analysis revealed the presence of 34 compounds with significant variability among regions. The seeds showed great antioxidant capacity with significant variability. Capparis spinosa seeds, due to their phenolic content and high antioxidant activity, could be a prospective source of natural bioactive molecules that could replace synthetic antioxidants.