Plantago major L. which is a medicinal plant with important biological activities, commonly used as traditional medicine. Potential inhibitory activities of the aqueous extract and three isolated constituents calceorioside B (1), homoplantaginin (hispidulin-7-O-glucoside) (2) and plantamajoside (3) from the aerial parts of Plantago major subsp. major L. (Plantaginaceae) have been tested against hyaluronidase, collagenase, and elastase, which play critical roles in wound pathogenesis. Even though, the extract (27.04%), and among the isolated compounds, calceorioside B (41.16%) exerted significant inhibition against hyaluronidase enzyme, homoplantaginin and plantamajoside were found to be inactive. Similar results were obtained from collagenase enzyme inhibition test. The extract (21.92%) and calceorioside B (28.34%) also caused notable inhibition in this test. However, no remarkable inhibition was observed in the presence of elastase enzyme. The experimental data revealed that P. major subsp. major displayed remarkable inhibitory activity against hyaluronidase and collagenase enzymes. In vitro enzyme activity of P. major subsp. major is reported for the first time in the current study. (C) 2019 The Author(s). Published by Elsevier B.V. on behalf of King Saud University.