Most of the existing chemotherapeutic drugs have plenty of side effects. Chinese herbal medicine has been used for pharmaceutical and dietary therapy for thousands of years with more effective and fewer side effects. Cestrum nocturnum (CN) has long been used to treat digestive diseases for centuries in China. Our previous study first proved that the n-butanol part isolated from the flowers of CN produced an inhibitory effect on the proliferation of malignant cells. However, the fractions responsible for the antiproliferation effect of n-butanol part from CN flowers and related mechanisms remain unknown. Thus, in this study, we extracted fractions C4 and C5 from n-butanol part of CN flowers and investigated their immune toxicity and antitumor activities. It was found that fractions C4 and C5 exhibited great cytotoxicity to cancer cell lines but had low immune toxicity towards T and B lymphocytes in vitro. The tested fractions also attenuated proliferation and induced apoptosis at G 0/G 1 and G 2/M phases in Bel-7404 cells through inducing DNA damage and inhibiting topoisomerase II relaxation activity. These results suggest that fractions C4 and C5 may represent important sources of potential antitumor agents due to their pronounced antitumor effects and low immune toxicity.