<jats:p>This work investigated the metabolites and their biosynthetic functional hydroxylase genes of the deep-sea sediment metagenomic clone 25D7. 5-Bromoindole was added to the 25D7 clone derived Escherichia coli fermentation broth. The new-generated metabolites and their biosynthetic byproducts were located through LC-MS, in which the isotope peaks of brominated products emerged. Two new brominated bis-indole metabolites, 5-bromometagenediindole B (1), and 5-bromometagenediindole C (2) were separated under the guidance of LC-MS. Their structures were elucidated on the basis of 1D and 2D NMR spectra (COSY, HSQC, and HMBC). The biosynthetic functional genes of the two new compounds were revealed through LC-MS and transposon mutagenesis analysis. 5-Bromometagenediindole B (1) also demonstrated moderately cytotoxic activity against MCF7, B16, CNE2, Bel7402, and HT1080 tumor cell lines in vitro.