Diabetes, a metabolic disorder, is caused by a high blood sugar level. Diabetes is an increasing health issue and search for potent antidiabetic agents is desirable. Owing to its ethnomedicinal value, the Himalayan perennial herb Bergenia stracheyi (Hook. f. & Thoms.) Engl. (Saxifragaceae Juss) is used to treat diabetes. Herein, an efficient high-speed countercurrent chromatography with elution mode is reported for separation of active compounds from B. stracheyi. In current investigation, six main compounds including β-arbutin (1), bergenin (2), 6-O-galloylarbutin (3), gallic acid (4), 11-O-galloylbergenin (5), and (-)-epicatechin 3-O-gallate (6) with above 95% purity were efficiently separated in a single run using biphasic tert-butyl methyl ether/n-butanol/methanol/water (1:3:1:5, v/v/v/v) solvent system. The structures of these compounds were characterized using spectral techniques and compared with the literature. Antidiabetic and antioxidant activities evaluation of the study samples showed that β-arbutin (1) and 6-O-galloylarbutin (3) have a significant protective effect, especially at high dose against hydrogen peroxide induced oxidative injury. Our results might help further in-depth phytochemical and biological evaluation studies in search of potent antidiabetic compounds from B. stracheyi.