© 2022 University of Ankara. All rights reserved.Objective: This study aimed to investigate the in vitro wound healing, anti-inflammatory, antimicrobial and antioxidant activity of Phlomis pungens Willd. extract derived from the aerial parts. Material and Method: The phytochemical analysis was performed using GC-MS in order to identify the volatile components of the bioactive Hex extract. The wound healing activity of P. pungens extract was evaluated based on in vitro antimicrobial, antioxidant, anti-inflammatory, and, scratch activity was studied. In addition, the in vitro cytotoxicity of the extract was also evaluated. Result and Discussion: P. pungens methanol extract depicted a 5-LOX inhibitory activity at 78.2µg/mL (IC50), while the antioxidant activity by DPPH radical provided an IC50=2.41mg/mL, and the ABTS radical showed IC50=3.32mg/mL, respectively. The extract showed dose-dependently anti-inflammatory activity while L-NAME and P. pungens methanol extract significantly decreased LPS stimulated PGE2 production. According to the scratch assay results, all treatments led to an increase in cell migration rate with a dose-dependent effect. Our findings suggested that P. pungens methanol extract may have a role in wound healing according to the scratch test, and it is thought that its antioxidant and anti-inflammatory activity also contributed. Further evaluations are ongoing to confirm the in vitro activity under in vivo conditions.