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Clerodendrum cyrtophyllum is a common medicinal plant used by Miao people. In order to study the chemical constituents and antibacterial activities of C. cyrtophyllum in Guizhou, China. Macroporous adsorption resin, reversed phase column chromatography, silica gel column chromatography, Sephadex LH-20 column chromatagraphy, and semi-preparative high performance liquid chromatography were used to separate and purify 95% ethanol extracts from C. Cyrtophyllum. The antibacterial activities were evaluated by MTT method. The results showed 18 compounds were isolated and identified as friedelin (1), α-amyrin palmitate (2), sambuculin A (3), 22-dehydroclerosterol (4), isopetasin (5), taraxasteryl palmitate (6), stigmasterol (7), 22-dehydroclerosterol-3-O-β-D-(6′-O-margaroyl)-glucopyranoside (8), 3-O-β-D-galactopyranosyl-(24β)-ethylcholesta-5,22,25-trien (9), cistanoside D (10), tortoside F (11), balanophonin (12), luteoloside (13), acteoside (14), luteolin (15), acantrifoside E (16), trichotomside B (17), cistanoside C (18). Among of them, compounds 2, 3, 5, 6, 8-13, 16-18 were all isolated from C. cyrtophyllum for the first time. Besides, biological activity test showed Compound 13 had antibacterial activity against Streptococcus uberis. The study enriches the chemical composition of C. cyrtophyllum, and provides reference for the drug development of anti-mastitis of dairy cows.