Flower extracts of Prunus spinosa L. (blackthorn)– a traditional medicinal plant of Central and Eastern Europe indicated for the treatment of urinary tract disorders, inflammation, and adjunctive therapy of cardiovascular diseases– were evaluated in terms of chemical composition, antioxidant activity, potential anti-inflammatory effects, and cellular safety in function of fractionated extraction. The UHPLC-PDA-ESI-MS3 fingerprinting led to full or partial identification of 57 marker constituents (36 new for the flowers), mostly flavonoids, A-type proanthocyanidins, and phenolic acids, and provided the basis for authentication and standardization of the flower extracts. With the contents up to 584.07 mg/g dry weight (dw), 490.63 mg/g dw, 109.43 mg/g dw, and 66.77 mg/g dw of total phenolics (TPC), flavonoids, proanthocyanidins, and phenolic acids, respectively, the extracts were proven to be rich sources of polyphenols. In chemical in vitro tests of antioxidant (DPPH, FRAP, TBARS) and enzyme (lipoxygenase and hyaluronidase) inhibitory activity, the extracts effects were profound, dose-, phenolic-, and extraction solvent-dependent. Moreover, at in vivo-relevant levels (1-5 µg/mL) the extracts effectively protected the human plasma components against peroxynitrite-induced damage (reduced the levels of oxidative stress biomarkers: 3-nitrotyrosine, lipid hydroperoxides, and thiobarbituric acid-reactive substances) and enhanced the total antioxidant status of plasma. The effects observed in biological models were in general dose- and TPC-dependent; only for protein nitration the relationships were not significant. Furthermore, in cytotoxicity tests, the extracts did not affect the viability of human peripheral blood mononuclear cells (PBMC), and might be regarded as safe. Among extracts, the defatted methanol-water (7:3, v/v) extract and its diethyl ether and ethyl acetate fractions appear to be the most advantageous for biological applications. As compared to the positive controls, activity of the extracts was favorable, which might be attributed to some synergic effects of their constituents. In conclusion, this research proves that the antioxidant and enzyme inhibitory capacity of phenolic fractions should be counted as one of the mechanisms behind the activity of the flowers reported by traditional medicine and demonstrates the potential of the extracts as alternative ingredients for functional products supporting the treatment of oxidative stress-related pathologies cross-linked with inflammatory changes, especially in cardiovascular protection.