Ptelea trifoliata L. is a perennial plant of the Rutaceae family and contains secondary metabolites with potential biological relevance. Due to limited information on its activity, the objective of this study was to evaluate the biological properties of its flower extracts and to determine their phytochemical composition. Flowers were dried and subjected to ultrasound-assisted extraction using methanol, 60% methanol and water. LC–MS/MS was used for qualitative profiling, HPLC for quantitative determination, and spectrophotometry for measuring total phenolic and flavonoid content. The antioxidative capacity of the extracts was determined using DPPH, CUPRAC, FRAP, and iron chelation assays. Enzymatic inhibition analyses were performed for hyaluronidase, indicative of anti-inflammatory properties, and tyrosinase, associated with pigmentation mechanisms. The wound-healing capacity was evaluated in vitro using a scratch assay. Our research revealed the highest levels of polyphenols in the 60% methanol extract and of flavonoids in the methanol extract. The occurrence of chlorogenic acid, rutin, hyperoside, and astragalin was also demonstrated. Both methanol and 60% methanol extracts demonstrated antioxidant effects. The methanol extract showed the greatest hyaluronidase inhibition, while the 60% methanol extract was the most effective in suppressing tyrosinase activity and promoting wound closure. Principal component analysis showed that the contents of polyphenols or flavonoids were associated with enzyme-inhibitory or antioxidant activities. Moreover, the 60% methanol and water extracts exhibited notable wound healing properties. These results highlight the antioxidant, enzyme-modulating and regenerative potential of P. trifoliata flower extracts, suggesting their possible use in biomedical and dermatological applications.