Romipeptides A and B ( 1 and 2), two new romidepsin derivatives, and three known compounds, chromopeptide A ( 3), romidepsin ( 4) and valine-leucine dipeptide ( 5) were isolated from the fermentation broth of Chromobacterium violaceum No. 968. Their structures were elucidated by interpretation of their UV, HR-ESI-MS and NMR spectra. The absolute configuration of compound 1 and 2 were established by single crystal X-ray diffraction analysis. Compounds 1–5 were evaluated for their anti-proliferative activities against three human cancer cell lines, SW620, HL60, and A549. The results showed most of these compounds exhibited antitumor activities in vitro, in which compound 2 displayed potent cytotoxicity to SW620, HL60 and A549 cell lines, with IC 50 of 12.5, 6.7 and 5.7 nmol·L −1, respectively.