Leucrocin I is an antibacterial peptide isolated from crocodile (Crocodylus siamensis) white blood cell extracts. Although Leucrocin I exhibits strong antibacterial activity, the hemolytic activity, which may compromise its therapeutic effects and the minimal inhibitory concentration (MIC) values of synthetic Leucrocin I seem higher than native peptide. To improve antibacterial analogues of Leucrocin I that lack hemolytic activity, we designed a Leucrocin I analogue molecule based on the Leucrocin I sequence. The peptide made from all D-amino acids was more active than the corresponding L-enantiomer. In our detailed study, the interaction between peptides and the cell membrane of Vibrio cholerae as part of their killing mechanism was studied by fluorescence and electron microscopy. The results show that the outer membrane was the target of action of Leucrocin I analogues. Finally, the cytotoxicity assays revealed that both L- and D-Leucrocin I analogues are non-toxic to mammalian cells. In order to expand the systematic approach to short-antibacterial peptide design, the principle of amino acid stretches tagged at the C terminal of Luecrocin I, which is an ultra-short antibacterial peptide, by tryptophan, arginine or lysine has been reported. The choice of amino acid type at each position of a stretch depends on the hydrophobic and hydrophilic regions visualized in the helical wheel pattern of Luecrocin I. The results suggest that this design by oligopeptide tagging should also consider the properties such as net charge, hydrophobicity, the content of hydrophobic amino acids, polar angle, the properly hydrophilic and hydrophobic facets. Amidation at C terminal and arginine substitute for lysine can increase selectivity between mammalian cells (hemolytic and MTT assay) and bacterial cells. KT2 and RT2 are effective against five bacterial strains tested (S. typhi DMST 22842, S. epidermidis ATCC 12228, E. coli ATCC 25922 and S. aureus ATCC 25923). They also have selectivity at their MICs, which were under approximately 10% hemolytic activity and no toxicity at 19 μM toward Vero and RAW 264.7 cells for KT2, whereas RT2 had no toxicity at 38 μM. The SEM images imply that the antibacterial mechanism of RT2, KT2, KT3 and CRT2 may depend on concentration rather than time. Circular dichroism results indicate that the main antibacterial mechanism of them might be DNA binding. Finally, RT2 and KT2 can be new alternative antibacterial agents or may be further developed for alternative antibiotics.