Plantago major L. and Plantago lagopus L. are cosmopolitan species, belonging to the Plantaginaceae family, used in traditional and modern medicine. In this study, a phytochemical evaluation of different aqueous and ethanolic extracts of leaves and roots of both species from the region of Beja in Tunisia was performed. Some biological activities, including antioxidant, anticancer and antibacterial were also done. LC-MS qualitative analysis revealed that the aqueous extracts of the roots of P. lagopus were richer in polyphenols, mainly flavonoids (Luteoline 7-rutinoside, Luteoline 7-rhamnoside) and hydroxycinnamic acids including caffeic acid, than the hydro-ethanolic extracts. Additionally, we identified for the first time the presence of salicylic acid in the hot aqueous extracts of roots of P. lagopus and its absence in the roots of P. major. The antioxidant activity of the extracts was assessed using cyclic voltammetry (CV), revealing that the voltammograms of leaf and root extracts from P. lagopus exhibited a higher antioxidant capacity compared to those of P. major. Antiproliferative activity, was determined against two-colon cancer cell lines, demonstrated that only the 12 h treatments with P. lagopus leaf and root aqueous and hydro-ethanolic extracts at low concentration were able to significantly reduce the colon carcinoma coli-2 (CaCo-2) cells proliferation. The antibacterial /antibiofilm activity was performed on yeast, Gram- negative and +positive bacterial strains. We demonstrated for the first time that ethanolic extracts of leaves and roots of P. lagopus have an inhibitory activity against Escherichia coli and Klebsiella pneumonia at MIC = 2 μg/mL for leaves and 4 μg/mL for roots.