Extensive use of classical antibiotics has led to the growing emergence of many resistant strains of pathogenic bacteria. Evidence has suggested that cationic antimicrobial peptides (AMPs) are of greatest potential to represent a new class of antibiotics. The largest group of AMPs comprises peptides that fold into an amphipathic alpha-helical conformation when interacting with the target microorganism. In the current study, a series of cationic AMPs of 20 amino acids was designed and synthesised based on four structural parameters, including charge, polar angle, hydrophobicity and hydrophobic moment. The effect of these parameters on antimicrobial activity and selectivity was assessed by structural and biological analyses. Our results indicated that high hydrophobicity and amphipathicity (hydrophobic moment) were correlated with increased haemolytic activity, whilst antimicrobial activity was found to be less dependent on these factors. Three of the synthetic AMPs (GW-Q4, GW-Q6 and GW-H1) showed higher antimicrobial activity and selectivity against a broad spectrum of Gram-positive and Gram-negative bacteria compared with the naturally occurring AMPs magainin 2a and pleurocidin. This study also demonstrates that these rationally designed cationic and amphipathic helical AMPs exhibited high selectivity against several Vibrio spp. and are potential agents for future use in the treatment of these marine pathogens.