Antimicrobial resistance poses a serious global health threat. To address this challenge, an urgent need for innovative strategies involving the development of new therapeutic modalities with unique modes of action has emerged. Over the past decade, antimicrobial peptides (AMPs) have shown great potency as a new class of antibiotics. Herein, we report the discovery of non-ribosomal peptides based on genome mining predictions of Streptomyces sp. H-KF8, a marine Actinomycete isolated from a remote Northern Chilean Patagonian fjord. Sequence engineering led to a serum-stable, non-cytotoxic qualified hit peptide active against gram-positive, gram-negative bacteria and yeast. Initial mode of action studies in uropathogenic E. coli suggest a dual mode of action with the peptides’ action not being limited to majorly affecting the cell membrane, but also inducing strong defect on DNA packing. Further investigations based on tandem mass tag (TMT) quantitative proteomics to identify differential expressed proteins (DEPs) in global protein expression of E. coli and fluorescence inhibition assays for DNA interaction were performed to elucidate the antibacterial mechanisms. These results indicate that the AMPs induce a widespread dysregulation of gene expression in E. coli, by strong and non-specific binding to DNA. This suggests that the peptides interfere with the bacterial transcriptional and translational machinery, as further supported from a cell-free in vitro transcription/translation (IVT) assay.