Products from Mentha species are widely used in medical applications, including wound healing, due to the bioactive compounds they may contain. However, data on the effect of Mentha longifolia L. on wound healing are limited. This study investigated the antimicrobial and wound healing-promoting effects of Mentha longifolia L. essential oil using in vitro methods. The chemical compositions of the essential oil were identified using gas chromatography/mass spectrometry, while the agar well diffusion and disk diffusion methods were used to determine antimicrobial activity against pathogenic strains. A scratch wound healing assay was performed following determination of the cytotoxic dose in human fibroblast cell lines. The media were used for biochemical analysis 48 h after the in vitro wound model. Immunohistochemical staining was applied to determine the contribution of the essential oil to wound healing. The essential oil exhibited varying levels of antimicrobial activity against the tested pathogens and increased cell viability. All doses in the scratch wound healing assay promoted wound closure in a shorter time than in the control group. TAC levels were higher in the treated groups than in the control group, while TOS, IL-6, and TNF-α levels were lower. Levels of expression of FGF 2, IGF, and TGF-β were higher in the treated groups than in the control group at increasing doses. The essential oil of Mentha longifolia L. exhibited antimicrobial effects and improved wound healing at doses of 5 µg/mL and 10 µg/mL.