Medicago minima is a pasture legume that grows almost all over the world. In Tunisia, it occupies various climatic environments and is considered the most abundant annual Medicago plant. However, this species is unconsumed and unused by humans. This study aimed to explore the phytochemical characteristics of Medicago minima selected from different provenances in Tunisia and subsequently investigate the influence of environmental factors on their phenolic composition and antioxidant activity. Therefore, a calorimetric method and DPPH tests provided the total phenolic and total flavonoid contents and antioxidant potential in roots, stems, leaves and seeds. High performance liquid chromatography (HPLC) identified and quantified four phenolic acids and three flavonoids in the studied organs. Roots and leaves showed the greatest phenolic compound content and had high antioxidant activity. Rutin and syringic acid (leaves) represent a characteristic for this species. For each organ, principal component analysis of phenolic profiles showed that the root's phenolic composition could be an indication of the plant adaptation to even small changes in its environments. Plants originating from a cold climate, higher altitude or semi-arid environment had the highest phenolic compound contents in their organs. Our findings provide useful information for the exploitation of the phenolic compounds in these weeds for the development of environmental sustainability.