Type 2 diabetes is a chronic multifactorial disease affecting more than 425 million people worldwide, and new selective α-glucosidase inhibitors with fewer side effects are urgently needed. In this study, a crude ethyl acetate extract of Machilus litseifolia was fractionated by solid-phase extraction using C₁₈ cartridges to give a fraction enriched in α-glucosidase inhibitors. Subsequent microfractionation and bioassaying of the eluate by high-performance liquid chromatography (HPLC) using a complementary pentafluorophenyl column allowed construction of a high-resolution α-glucosidase inhibition profile (biochromatogram). This was used to target high-performance liquid chromatography – photodiode array detection – high-resolution mass spectrometry – solid-phase extraction – nuclear magnetic resonance spectroscopy (HPLC-PDA-HRMS-SPE-NMR) analysis towards α-glucosidase inhibitors. This led to the identification of 13 di-coumaroylated flavonol rhamnosides, of which seven (8, 10, 12a, 12b, 16, 17, and 18) are reported for the first time, and two lignans, of which one (5) is reported for the first time. IC₅₀ values of isolated compounds towards α-glucosidase range from 5.9 to 35.3 μM, which is 8 to 91 times lower than the IC₅₀ value of 266 μM measured for the reference compound acarbose.