Centaurea depressa M. Bieb. is used in traditional medicine as wound healing, antidiabetic, antibacterial, antimalarial, antirheumatic, diuretic, antipyretic, antibacterial, tonic, astringent, choleretic. It is popularly used as poultice, pounded, infusion, decoction, external, and internal. This study aimed to develop and in vitro characterize the methanol extract of Centaurea depressa (MCD)-containing nanoemulsion formulation (MCD-NEF) to heal the wound effectively and analyze the phenolic compounds. MCD-NEF was prepared and its droplet size, PDI, and ZP, and viscosity were determined. The effect of MCD and MCD-NEF on human dermal fibroblast cell viability was examined by MTT assay. MCD's phenolic compound profiles were analyzed by LC-MS/MS. Also, MCD and MCD-NEF's wound healing potential was evaluated using the scratch assay. The droplet size, PDI, ZP, pH and viscosity values for the MCD-NEF were found to be 152.42 ± 3.50 nm, 0.187 ± 0.020, (−)27.67 ± 1.68 mV, 5.57 ± 0.03, and 1.891 ± 0.010 cP, respectively. In the cell viability assay, the highest percentage of living cells was obtained at concentration of 16 μg/mL for the MCD and MCD-NEF. In the scratch assay, MCD-NEF showed significantly ameliorated wound closure % rates at 24 and 30 h than the control, MCD and blank-NEF (without MCD) groups (p < 0.001). In this study, immunofluorescence staining was carried out. TGF-β1 and FGFR-2 expressions in MCD-NEF-treated fibroblast cells increased significantly at 24 and 30 h compared to the control, MCD, and B-NEF groups (p < 0.001). MCD's. Twenty-seven phenolic compounds, including quinic acid, chlorogenic acid, cynaroside, cosmosiin, protocatechuic acid and isoquercitrin were determined by LC-MS/MS to be major phytochemical compounds in MCD. Our findings show that MCD-NEF may be promising for better wound healing.