Dithiolopyrrolones are microbial natural products containing a disulfide or thiosulfonate bridge embedded in a unique bicyclic structure. By interfering with zinc ion homeostasis in living cells, they show strong antibacterial activity against a variety of bacterial pathogens, as well as potent cytotoxicity against human cancer cells. In the current study, two new dithiolopyrrolones, pyrroloformamide C (<b>3</b>) and pyrroloformamide D (<b>4</b>), were isolated from <i>Streptomyces</i> sp. CB02980, together with the known pyrroloformamides <b>1</b> and <b>2</b>. The biosynthetic gene cluster for pyrroloformamides was identified from <i>Streptomyces</i> sp. CB02980, which shared high sequence similarity with those of dithiolopyrrolones, including holomycin and thiolutin. Gene replacement of <i>pyfE</i>, which encodes a nonribosomal peptide synthetase (NRPS), abolished the production of <b>1</b>-<b>4</b>. Overexpression of <i>pyfN</i>, a type II thioesterase gene, increased the production of <b>1</b> and <b>2</b>. Genome neighborhood network analysis of the characterized and orphan gene clusters of dithiolopyrrolones revealed a unified mechanism for their biosynthesis, involving an iterative-acting NRPS and a set of conserved tailoring enzymes for the bicyclic core formation.