Increased resistance to conventional antibiotics has become a major problem worldwide. Existing antibiotics are increasingly ineffective, as a result of resistance, becoming imperative to find new antibacterial strategies. One of the most promising possibilities is the use of antimicrobial peptides (AMPs), which play an important role in the innate immune response of different organisms. Some of these peptides can also act against cancer cells (anticancer peptides, ACPs). AMPs are small cationic amphipathic molecules. Their mechanisms of action are not fully understood, but their physicochemical properties are determinant, namely their amphipathic conformation upon interaction with biomembranes and their positive net charge, which allows them to interact preferentially with negatively charged biomembranes, like those of Gram-negative bacteria. Until now, AMPs demonstrate a low propensity for drug resistance development.Two different AMPs (Pa-MAP 1.5 and 1.9) were synthetically created based on an antifreezing peptide from the Antarctic fish Pleuronectes americanus. Both peptides showed promising therapeutic results against bacteria and cancer cells. Membrane leakage tests using lipid vesicles to mimic bacteria and cancer cells have shown that these two AMPs efficiently induce membrane disruption. Dynamic light scattering, surface plasmon resonance and zeta-potential studies confirmed the interaction of the peptides with the membranes. Studies using fluorescent probes that report different aspects of peptide-membrane interaction (DPH, TMA-DPH, di-8-ANEPPS and Laurdan) were also conducted, either using model vesicles or with Escherichia coli. Atomic force microscopy imaging of the AMPs effects on live cells and flow cytometry measurements confirmed the results obtained with lipid vesicles. The results obtained show that, although these two AMPs have several similarities, they act through different mechanisms of action.