In the present work, fourteen cultivars of Prunus domestica were analyzed to investigate their phenolic pattern with the purpose of using the leaves as potential resources of bioactive compounds in the pharmaceutical and food industry. Microwave-assisted extraction (MAE), dispersive liquid–liquid microextraction and sugaring-out liquid–liquid extraction techniques were optimized in order to obtain an exhaustive multi-component panel of phenolic compounds. The best phenolic-enriched recovery was achieved using MAE in water:methanol (30:70), and this procedure was further applied for quantitative analysis of phenolic compounds in real samples. In order to prove the safeness of these extracts, the biological potential of the Prunus cultivars was tested by several in vitro antioxidant and enzyme inhibitory assays. Moreover, their cytotoxicity was evaluated on human gingival fibroblasts (HGFs), and in most of the cases the treatment with different concentrations of extracts didn't show cytotoxicity up to 500 μg/mL. Only ‘Carpatin’ and ‘Minerva’ cultivars, at 250 and 500 μg/mL, reduced partially cell viability of HGFs population. Noteworthy, Centenar cultivar was the most active for the α-glucosidase inhibition (6.77 mmolACAE/g extract), whereas Ialomița cultivar showed the best antityrosinase activity (23.07 mgKAE/g extract). Overall, leaves of P. domestica represent a rich alternative source of bioactive compounds. •Determination of phenolic compounds in cultivars of Prunus domestica dry leaves.•Optimization of DLLME, SULLE, MAE of 14 cultivars of Prunus domestica.•Cytotoxicity of each extract against human gingival fibroblasts was performed.•Enzyme inhibitory assays were performed on extracts of Prunus domestica leaves.