Antimicrobial resistance is a serious and global health threat. To combat antimicrobial resistance new strategies involving the development of new modalities with a unique mode of action are urgently needed. Antimicrobial peptides (AMPs) have shown great potency as a new class of antibiotics in the last few years. 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. Based on these predictions, a series of eight peptides, including also cyclic peptides were designed and chemically synthesized. Our results show that though sequentially similar peptides show different microbicidal and structural properties. Microbicidal effect was observed against gram-negative, gram-positive bacteria and yeast. To understand their structure-activity relationships, characterization of the secondary structure and mode of action studies were performed. Mode of action studies suggests that these peptides potentially act on the cell membrane via a novel mechanism allowing the passage of small ions resulting in the dissipation of the membrane potential. The peptides’ structural similarity, which was determined by NMR spectroscopy, allowed us to derive a preliminary lead structure against gram-negative bacteria, which can serve as a basis for more potent cAMPs in future studies.