Background <p><i>Plantago major</i> subsp. <i>intermedia</i> has been traditionally used in Türkiye for the treatment of wounds, abscesses, constipation, pain, ulcers, hemorrhoids and gynecological diseases. In the present study, the phytochemical composition of <i>P. major</i> subsp. <i>intermedia</i> (Gilib.) Lange was investigated and its anti-inflammatory and wound healing effects were examined in light of existing experimental evidence from related <i>Plantago</i> species.</p> Methods <p>MeOH, 80% EtOH and water extracts of the plant were prepared. In vivo anti-inflammatory effects of these extracts were investigated using an acetic acid-induced capillary permeability model and their wound healing effects were studied using linear incision and circular excision wound models. Isolation studies were performed on the 80% EtOH extract. Furthermore, in vitro studies were conducted on the 80% EtOH extract, the main fractions obtained from this extract and the some of the isolated pure compounds. In these in vitro studies, anti-inflammatory effects were investigated by measuring nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-<i>α</i>) levels and wound healing effects were investigated using the scratch test.</p> Results <p>A total of six compounds were isolated from <i>P. major</i> subsp. <i>intermedia</i>: Isotachioside (1), aucubin (2), 10-hydroxymajoroside (3), 10-acetoxymajoroside (4), martynoside (5) and acteoside (6). All tested compounds significantly inhibited NO production at concentrations of 25, 50 and 100 µM (12.68%-61.89%). Although in vivo results were not statistically significant, the 80% EtOH extract showed the highest efficacy in linear incision and circular excision wound models.</p> Conclusions <p>The results of this study indicate the in vitro anti-inflammatory and wound healing activities of <i>P. major</i> subsp. <i>intermedia</i> and are consistent with previously reported biological activities of <i>P. major</i>.</p>