Objective: To date, polychaetes remain a poorly studied class of invertebrate animals in terms of the features of functioning of their immune system and, in particular, the biodiversity of antimicrobial peptides (AMPs). AMPs also known as host defense peptides play a key role in host protection from various pathogens and regulation of the species composition of symbiotic microbes. A study of the biosynthesis of AMPs in polychaetes resulted in the discovery of the so-called BRICHOS domain in the structure of the precursor proteins of a number of such peptides. The conserved structure of this domain makes possible the bioinformatic search for AMP precursors in polychaete transcriptomes. In this work, we found and studied a novel BRICHOS-related AMP from the lugworm Arenicola marina, representing a previously undiscovered in polychaetes a structural family of defensin-like peptides stabilized by four disulfide bonds. Methods: The peptide, designated as AmBRI-44a and containing 44 amino acid residues, was obtained by heterologous expression in Escherichia coli. The peptide secondary structure was investigated by CD spectroscopy in water and dodecylphosphocholine (DPC) micelles. The minimum inhibitory concentrations (MICs) against a wide range of bacterial pathogens were assessed using the two-fold serial dilutions method. Cytotoxicity of AmBRI-44a was studied in vitro on human erythrocytes or adherent cell line HEK293T using the hemoglobin release assay or the MTT test, respectively. The AMBRI-44a potential target was discovered by successive daily subculturing of the AmBRI-44a resistant strain followed by whole-genome sequencing. Results and Discussion: According to CD data, AmBRI-44a is a predominantly β-structured peptide. AmBRI-44a was shown to have a specific activity against a narrow spectrum of Gram-positive bacteria and pronounced cytotoxic effects on the eukaryotic cell line HEK293T. The proposed mechanism of the antibacterial action of this peptide is associated with the inhibition of bacterial cell wall biosynthesis, as indicated by the genetic and phenotypic analysis of selected AmBRI-44a-resistant bacteria Bacillus licheniformis B-511. Conclusions: The resulting data allow us to consider the discovered peptide AmBRI-44a as a candidate compound for the development of an antibiotic agent that could potentially be effective in the treatment of infectious diseases mediated by multidrug-resistant Gram-positive bacteria.