This study investigates the potential of antimicrobial peptides (AMPs) as alternatives to combat antibiotic resistance, with a focus on two AMPs containing unnatural amino acids (UAAs), E2-53R (16 AAs) and LE-54R (14 AAs). In both peptides, valine is replaced by norvaline (Nva), and tryptophan is replaced by 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic). Microbiological studies reveal their potent activity against both Gram-negative (G(-)) and Gram-positive (G(+)) bacteria without any toxicity to eukaryotic cells at test concentrations up to 32 μM. Circular dichroism (CD) spectroscopy indicates that these peptides maintain α-helical structures when interacting with G(-) and G(+) lipid model membranes (LMMs), a feature linked to their efficacy. X-ray diffuse scattering (XDS) demonstrates a softening of G(-), G(+) and eukaryotic (Euk33) LMMs and a nonmonotonic decrease in chain order as a potential determinant for bacterial membrane destabilization. Additionally, XDS finds a significant link between both peptides' interfacial location in G(-) and G(+) LMMs and their efficacy. Neutron reflectometry (NR) confirms the AMP locations determined using XDS. Lack of toxicity in eukaryotic cells may be related to their loss of α-helicity and their hydrocarbon location in Euk33 LMMs. Both AMPs with UAAs offer a novel strategy to wipe out antibiotic-resistant strains while maintaining human cells. These findings are compared with previously published data on E2-35, which consists of the natural amino acids arginine, tryptophan, and valine.