Euphorbia humifusa Willd. has been used in traditional Chinese medicine. Herein we report the isolation of its constituents and the testing results of their antimicrobial and antitumor activities. The results show that the crude petroleum ether, chloroform, ethyl acetate, butanol and residual methanol fractions exert such activities. Particularly, the crude petroleum ether and ethyl acetate fractions exhibit antigrowth activity against Staphyloccocus aureus with MIC 0.5 mg/mL. Petroleum ether and chloroform fractions significantly exhibit potent antigrowth activity against Caco-2 cells, with IC50 value of 0.059 and 0.025 mg/mL, respectively. The promising bioactivities of the crude petroleum ether fractions guided the first isolation of compounds: 24-methylene cycloartanone (2), dibutyl phthalate (3), hexacosanol (4) and bis-(2-ethylhexyl) phthalate (5) from this species. The antimicrobial activities of compounds 1-2 were evaluated. The tested compounds exhibit potent antigrowth activity against Staphyloccocus aureus, Escherichia coli and Canidia albicans.