Antimicrobial peptides (AMPs) are essential components of the innate immune response, which play a significant role in combating pathogenic infections. Hepcidin, a peptide hormone predominantly synthesized in the liver, has been identified to exhibit dual functions in iron metabolism and antimicrobial activity across various organisms. In this study, we describe the molecular characteristics, anti-vibrio activity, and mechanisms of action of a novel hepcidin isoform from the commercially important estuarine fish, Whipfin silver-biddy (Gerres filamentosus). The open reading frame of hepcidin cDNA sequence was 273 base pairs in length, encoding a peptide of 90 amino acids. The active region Gf-Hep contains eight well-conserved cysteine residues which form disulfide bridges stabilizing the antiparallel beta sheet conformation of the peptide. Featuring a C-terminal furin cleavage site (RXXR) within the prodomain and an N-terminal 'QSHI/LS' motif in the mature region, Gf-Hep is classified with the HAMP1 group of fish hepcidins. Recombinantly expressed Gf-Hep exhibited robust antimicrobial activity against Vibrio parahaemolyticus, Vibrio fluvialis, Vibrio cholerae, and Vibrio alginolyticus. The modes of action of rGf-Hep included membrane depolarization, membrane permeabilization, and ROS production. With its potent antibacterial properties, direct killing mechanisms, and non-cytotoxic effects on normal cells, rGf-Hep holds promise to be developed as an effective anti-vibrio agent for aquaculture applications.