Seven new stilbene glycosides including three dimers (1−3) and four monomers (4−7) were isolated from the roots of Polygonum multiflorum along with nine previously identified stilbenes (8−16). In addition, two deglucosylated stilbenes, 2a and 3a, were also obtained as new dimeric stilbenes. The structures of the purified phytochemicals were elucidated by interpreting their spectroscopic data (NMR, HRMS, and ECD). To the best of our knowledge, this represents the first isolation of a phenylpropanoid (C 6 −C 3 ) substituted with a stilbene unit (7) from the Polygonaceae family. In an in vitro enzyme assay with human recombinant protein tyrosine phosphatase-1B (PTP1B), compounds 2−5 showed weak PTP1B inhibition with an IC 50 value range of 27.4−37.6 μM, while three deglucosylated stilbenes 2a, 3a, and 8a exhibited IC 50 values of 2.1, 1.9, and 12.1 μM, respectively. The inhibition modes and binding mechanism of selected inhibitors (2a and 3a) were investigated using kinetic methods and molecular docking simulations.