Dithiolopyrrolones are microbial natural products containing a disulfide or thiosulfonate bridge embedded in a unique bicyclic structure. In the current study, two new dithiolopyrrolones, pyrroloformamide C (3) and pyrroloformamide D (4), were isolated from Streptomyces sp. CB02980, together with the known pyrroloformamides 1 and 2. The biosynthetic gene cluster for pyrroloformamides was identified from S. sp. CB02980, which shared high sequence similarity with those of dithiolopyrrolones, including holomycin and thiolutin. Gene replacement of pyfE, which encodes a non-ribosomal peptide synthetase, abolished the production of 1－4. Overexpression of pyfN, a type II thioesterase gene, increased the production of 1 and 2. The structure elucidation and biosynthetic characterization of pyrroloformamides 1 - 4 may inspire future efforts to discover new dithiolopyrrolones, which are promising drug leads for the treatment of infectious diseases or cancer.