The effect of the antimicrobial tetradecapeptide Polybia-MP1 on the dynamics, viscosity, and bending of the host membrane has been investigated using different membrane compositions. Membranes composed of pure POPC, and binary mixtures with cholesterol (Chol) or the hopanoid diplopterol (Dip) were selected, and the interaction of Polybia-MP1 with lipid monolayers and bilayers (GUVs and LUVs) was studied. Hopanoids are proposed sterol-surrogates in sterol-lacking bacteria. The inclusion into POPC/Chol occurs in a lesser extent and inducing less disorder than in the other membranes. This is intriguing because Dip has been proved to induce similar order than chol in model membranes and in bacteria. Despite the hopanoid, MP1 induced similar dye release from LUVs of POPC and POPC/Dip. The effect of the peptide on the membrane flexibility was investigated by generating nanotubes from single GUVs using optical tweezers. Analysis of the nanotube refraction showed that the membrane rigidity decreased with time and in the following order POPC