To study the chemical constituents of Stephania yunnanensis H. S. Lo and their in vitro antibiotic activities. Silica gel, alumina and Sephadex LH-20 chromatographic methods are used to isolate alkaloidal constituents. Structures of the compounds are identified based on physicochemical properties and spectral analysis. Kirby-Bauer test and porous plate method are used to study the inhibitory activities of isolated chemical constituents on methicillin-resistant S. aureus (MRSA). Five chemical constituents are isolated from the tuber extract of Stephania yunnanensis H. S. Lo, namely tetrahydropalmatine (1), palmatine (2), sinoacutine (3), dicentrine (4) and jatrorrhizine (5). Inhibition zone diameters of tetrahydropalmatine and palmatine against MRSA are larger at a concentration of 50 mg/mL, which are 13 mm and 16 mm, respectively; tetrahydropalmatine and palmatine both have inhibitory effects on MRSA, with MIC of 0.312 μg/ml and 0.156 μg/ml. In the porous plate method, IC50 of tetrahydropalmatine and palmatine are 0.436 μg/ml and 0.201 μg/ml. Alkaloidal constituents in Stephania yunnanensis H. S. Lo are mainly berberine type, morphinan type and protoberberine type alkaloids. Besides, tetrahydropalmatine and palmatine have rather significant anti-MRSA activities.