Potentilla L. species have a long history of use in folk medicine for the management of various human ailments. Despite the tremendous number of studies describing the phytochemical and pharmacological profile of a number of Potentilla species, there is a paucity of scientific data regarding the biological activity of Potentilla norvegica. Thus, the present study endeavored to assess the total phenolic content (TPC), total flavonoid content (TFC), and secondary metabolite composition of this plant using LC-ESI-MS-TOF analysis and determined the biological potential of P. norvegica extracts and fractions. Assessment of the antioxidant potential was performed in vitro by using five standard assays (FRAP, CUPRAC, ABTS, DPPH, and metal chelation assays). Moreover, the inhibition of selected enzymes (α-amylase, α-glucosidase, acetylcholinesterase (AChE), butyrylcholinesterase (BChE), tyrosinase) was examined. During the study, the following extracts and fractions of aerial parts of P. norvegica were analyzed: water (PN1), 50% MeOH (PN2), MeOH (PN3), Et2O (PN4), EtOAc (PN5), and n-BuOH (PN6). The highest TPC and TFC were found in the PN4 fraction (486.21 ± 14.58 mg gallic acid equivalents (GAE)/g extract and 24.22 ± 0.93 mg rutin equivalents (RE)/g extract, respectively), which may be attributed to the highest ABTS, CUPRAC and metal chelation activities of this fraction (with values of 3.01 ± 0.01, 5.90 ± 0.73 mmol Trolox equivalents (TE)/g extract and 20.15 ± 0.11 mg EDTA equivalents (EDTAE)/g extract, respectively). Furthermore, the PN5 fraction was found to be the most effective in the phosphomolybdenum, DPPH and FRAP assays (5.06 ± 0.02, 1.55 ± 0.01 and 5.19 ± 0.12 mmol TE/g extract, respectively). PN2 exhibited the highest inhibitory potential against AChE and BChE (2.58 ± 0.01 and 2.31 ± 0.13 mg galantamine equivalents (GALAE)/g extract, respectively), which is probably related to the more complete fingerprint of the analyzed extract. Tyrosinase was inhibited most potently by the PN1 extract (50.33 ± 1.63 mg kojic acid equivalents (KAE)/g extract). However, all the extracts and fractions exerted mild activity against α-glucosidase and α-amylase (the highest values were 2.03 ± 0.02 and 0.30 ± 0.03 mmol acarbose equivalents (ACE)/g extract for PN4 and PN3, respectively). Furthermore, LC-ESI-MS analysis revealed the predominant presence of polyphenolics, with the presence of acacetin indicated in the Potentilla genus for the second time. These observations highlighted the potential applications of P. norvegica as a valuable source of antioxidants, with interesting activities for the development of new treatment strategies against several human diseases in the pharmaceutical sector.