In order to discover new lead compounds targeting DNA or DNA topoisomerases, we screened cultures of actinomycetes and fungi using an assay with Bacillus stearothermophilus NUB3620 and bacteriophage TP-68 (to detect compounds inhibiting phage growth without antibacterial activity against NUB3620), and identified strain KYI1768 (Streptomyces sp.) producing novel pyrrole-amide antitumor antibiotics, pyrronamycin A and B. We described their production (fermentation at 25°C for 42 hours in a 30-liter jar fermenter, isolation via Diaion HP-20/HP-20SS columns and HPLC purification to obtain water-soluble white solids)、structure (containing pyrrole-amide repeating unit, elucidated by ¹H, ¹³C and ¹⁵N NMR with ¹⁵N-labeled pyrronamycin B) and biological activities: both showed antibacteriophage activity comparable to each other (distamycin A was inactive up to 10μg/disc), did not affect mammalian topoisomerase I or II in vitro and in cells, induced dose-dependent band shift of supercoiled pBR322 DNA to form stable drug-DNA complexes (unlike distamycin A's reversible binding), exhibited potent antimicrobial activity against Gram-positive (e.g., Bacillus subtilis, Staphylococcus aureus) and Gram-negative bacteria (e.g., Escherichia coli, Klebsiella pneumoniae) with MIC values superior to distamycin A, had cytotoxic activity against HeLaS3 cells (IC50: 1.2μM for A, 1.0μM for B; distamycin A IC50 >100μM), and pyrronamycin B showed in vivo antitumor activity against sarcoma 180 (T/C 0.44 at 0.38mg/kg) and human lung carcinoma A549 (T/C 0.51 at 0.25mg/kg) in nude mice. Stable DNA complex formation may contribute to their superior biological activity over distamycin A. Further studies on the mode of DNA binding and in vivo antitumor activity are underway.