Biomimetic membranes are used extensively for the study of peptide-membrane interactions, especially the mechanism of action of membrane disrupting antimicrobial peptides. Much attention was paid to the role of, and potential artefacts introduced by, the composition of these membranes; however the physical state of the membrane, i.e. whether it is in a vesicular, unilamellar or multilamellar form has been treated largely irrelevant. Yet, such different model membranes have different physicochemical characteristics in terms of membrane tension, lipid mobility and distribution etc. Here we show that these differences can be identified in the data gained about peptide-lipid interactions. Quartz crystal microbalance with dissipation (QCM) -based nanoviscosity measurements were used to investigate the interaction mechanism of the well known membrane disrupting peptide Mellitin with three different supported model systems: highly vesicular layers, multiple bilayer stacks and single bilayer membranes. We demonstrate that the different models yield slightly but noticeably different results, and hence the interpretation of the data for peptide-membrane interaction mechanism has to factor in the physical state of the membrane. It is also apparent from the data that the mechanism of action of the peptide itself is affected by the membrane structure.