The genus Daphne presents great interest as a valuable source of natural agents in traditional medicine of various countries, including Turkey. In the current work, we investigated the phytochemical composition and biological properties of Daphne pontica L. extracts. Methanol extracts of roots, stems and leaves were prepared. The chemical characterization was performed by HPLC-ESI-MSn, observing that flavonoids and phenolic acids were the main compounds. Biological properties were studied in terms of antimicrobial, DNA protection, anticancer, and enzyme inhibitory properties (cholinesterases, tyrosinase, amylase and glucosidase). Methanol extracts of roots, leaves, and stems of D. pontica had antimicrobial effects against a broad range of bacteria. In addition, methanol extract of roots of D. pontica exhibited a promising anticancer activity that was shown to be dose- and time-dependent for HeLa cells with an IC50 value of 203.9 μg/mL. Generally, the leaf extracts exhibited the best enzyme inhibitory properties on the tested enzymes. According to our results, D. pontica might be regarded as a valuable source of natural agents to combat cancer, infectious diseases, and metabolic diseases.