Prunus mahaleb has garnered attention as a potent medicinal agent and functional component. We aimed to detect the chemical composition and biological activities of several parts (fruit, leaves, and twigs) of P. mahaleb. Biological activities were assessed for antioxidant properties, enzyme inhibition, mutagenic/antimutagenic effects, and antibacterial efficacy. Antioxidant capabilities were evaluated using various assays, including DPPH, ABTS, CUPRAC, FRAP, phosphomolybdenum, and metal chelating. The chemical constituents of the extracts were identified and quantified using the HPLC-ESI-MS/MS method. The effects of enzyme inhibition were examined on some enzymes, including AChE, BChE, tyrosinase, amylase, and glucosidase. The Ames test was used to evaluate the mutagenic and antimutagenic properties of the plant extracts. Furthermore, a broth microdilution assay was employed to evaluate the possible antibacterial effects of the extracts against microorganisms. The methanol extract of twigs showed superior antioxidant capabilities (DPPH: 388.39 mg TE/g; ABTS: 701.50 mg TE/g; CUPRAC: 459.05 mg TE/g; FRAP: 264.99 mg TE/g). The methanol extract of twigs demonstrated the highest tyrosinase inhibitory activity (61.91 mg KAE/g). A total of 40 metabolites, mainly flavonoids, were detected through HPLC-ESI-MS/MS analysis, revealing that ferulic acid, naringenin, and herniarin were the predominant compounds. In the Ames test, the tested extracts exhibited no mutagenic potential. The antimutagenicity assay demonstrated that methanol and ethyl acetate extracts from twigs and leaves were particularly efficient against frameshift and base pair substitution mutations induced by recognized mutagens. The metabolic activation system amplified these strong activities to inhibition rates ranging from 85% to 98%. The results from the antibacterial assay indicated antibacterial effectiveness at dosages between 6.25 and 0.195 mg/mL, particularly effective against Sarcina lutea, Bacillus cereus, Candida albicans, and Staphylococcus aureus. Our findings indicate that P. mahaleb can serve as a versatile raw material for the development of health-promoting applications, including medicines, cosmeceuticals, and nutraceuticals.