Abstract The cathelicidin-derived antimicrobial tritrpticin could be classified as either Trp-rich or Pro/Arg-rich peptide. We recently found that the sequence modification of tritrpticin focused on Trp and Pro residues led to considerable change in structure and antimicrobial potency and selectivity, but their mechanisms of microbial killing action were still unclear. Here, to better understand the bactericidal mechanisms of tritrpticin and its two analogs, TPA and TWF, we studied their effect on the viability of Gram-positive S. aureus and Gram-negative E. coli in relation to their membrane depolarization. Although TWF more effectively inhibited growth of S. aureus and E. coli than TPA, only a 30 min exposure to TPA was sufficient to kill both bacteria and TWF required a lag period of about 3–6 h for bactericidal activity. Their different bactericidal kinetics was associated with membrane permeabilization, i.e., TWF showed negligible ability to depolarize the cytoplasmic membrane potential of target cell membrane, whereas we observed significant membrane depolarization for TPA. In addition, while TPA caused rapid and large dye leakage from negatively charged model vesicles, TWF showed very little membrane-disrupting activity. Interestingly, we have looked for a synergism among the three peptides against E. coli, supporting that they are working with different modes of action. Collectively, our results suggest that TPA disrupts the ion gradients across the membrane, causing depolarization and a loss of microbial viability. By contrast, TWF more likely translocates across the cytoplasmic membrane without depolarization and then acts against one or more intracellular targets. Tritrpticin exhibits intermediate properties and appears to act via membrane depolarization coupled to secondary intracellular targeting.