Medicinal plants have been widely used as (poly)pharmacological remedies and constitute a rich source for antidiabetic drug discovery. In the present study, forty medicinal plant samples collected in China were tested for inhibitory activity against α-glucosidase, α-amylase, and protein-tyrosine phosphatase 1B (PTP1B). Crude ethyl acetate extracts of Dioscorea bulbifera L., Boehmeria nivea Gaudich, Tinospora sagittata Gagnep. and Persicaria bistorta (L.) Samp. showed dual inhibitory activity towards α-glucosidase and PTP1B, and were chosen for further investigation. Subsequent dual high-resolution α-glucosidase/PTP1B profiling or triple high-resolution
α-glucosidase/α-amylase/PTP1B profiling combined with HPLC-HRMS and NMR spectroscopy led to the identification of 28 metabolites with one or more bioactivities. Among these, three new phenanthrenes were identified from D. bulbifera, including one new biphenanthrene (10) exhibiting promising dual inhibitory activity towards α-glucosidase and PTP1B with IC₅₀ values of 2.08 ± 0.19 and 3.36 ± 0.25 μM, respectively. Two triterpenoids and one fatty acid from B. nivea and T. sagittata as well as some commercially available fatty acids showed strong PTP1B inhibitory activity with IC₅₀ values in the range of 4.89 ± 0.38 to 53.77 ± 4.20 μM.