Halocidin is a heterodimer antimicrobial peptide previously isolated from the tunicate Halocynthia aurantium. Based on the larger monomer (18Hc) of halocidin, nine halocidin congeners, including a series of 6 peptides truncated successively from the carboxyl-terminal end of 18Hc and 3 analogs (18HcKK, K19Hc, and K19HcKK), which have lysine residues in place of two internal histidines or have a lysine added to the amino terminus of the 18Hc molecule, were prepared. Each peptide was also converted into a homodimeric version. The antimicrobial activities of halocidin congeners truncated from the C terminus were dramatically decreased, suggesting that the full length of 18Hc is required for maintaining its maximum antimicrobial activity. Dimer forms of halocidin congeners exhibited stronger antimicrobial activities than the monomer of the corresponding peptide. Four dimer peptides (di-18Hc, di-18HcKK, di-K19Hc, and di-K19HcKK) were analyzed for antimicrobial activities against 10 clinically isolated antibiotic-resistant bacteria in elevated concentrations of NaCl or MgCl2. Of the peptides studied here, di-K19Hc retained invariably strong activity against all bacteria in diverse conditions and also showed much reduced hemolytic activity against human erythrocytes.