The objective of this study is to measure and compare the effects of the cell penetrating peptide (CPP) Pep-1 and the antimicrobial peptide (AMP) combi-2 on vesicles of membranes mimicking Escherichia coli (E. coli). To characterize the effects of Pep-1 and combi-2 on E. coli membrane vesicles, a combination of five biophysical techniques was employed: fluorescence, infrared, scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC) techniques. Upon addition of E. coli membranes, tryptophan fluorescence intensity of Pep-1 showed a sudden blue-shift and decreased in a nonconcentration-dependent manner while the intensity of combi-2 decreased in a concentration-dependent manner, most significantly for a very low peptide-to-lipid ratio of 1:40. Complexes of Pep-1 and combi-2 with E. coli membrane mimicking vesicles having shown a significant blue-shift in fluorescence intensity were then prepared and studied in freeze-dried states. IR results indicate that Pep-1 and combi-2 adopt a major 3(10)-helix structure in the presence of E. coli membrane mimicking vesicles at low peptide concentration. Pep-1 and combi-2 have a similar effect on E. colt membrane mimicking vesicles at low concentration even though combi-2 is in the interfacial region of the bilayer while Pep-1 is located between the interfacial region and the hydrophobic region. Combi-2 at low concentration acts as a CPP. TGA and DSC results reveal that combi-2 has a stabilizing effect on E. coli at any concentration while Pep-1 stabilizes the E. coli membrane only at high concentration. Both peptides show a preferential interaction with one of the anionic lipids leading to clustering in E. coli membrane. SEM images reveal that Pep-1 and combi-2 form superstructures including fibrils in the presence of E. coli membrane mimicking vesicles. Calorimetric and spectroscopic techniques may be used in a complementary way with imaging techniques to gain more insights into peptide-lipid interactions.