Abstract Steady-state fluorescence anisotropy and dynamic light scattering (DLS) were used to determine the thermotropic properties of lipid systems that act as models for bacterial membranes of Yersinia kristensenii and Proteus mirabilis. Lipid proportions of PE:PG:CL of 0.60:0.20:0.20 and 0.80:0.15:0.05, were used in order to mimic these two membranes respectively. We observed that the introduction of cardiolipin (CL) as a third lipid component of any PE:PG mixture, changes the system's properties considerably. The results obtained by these two techniques show that the main transition temperatures obtained are undoubtedly CL-dependent. Additionally AFM experiments were performed and these results show that even at small concentration CL produces important changes not only in the membrane thermotropic properties, but also in the bilayer structure. In summary, we were able to compare how low and high CL concentration affect bacterial membrane model system properties which can provide a further explanation for the different antibiotic susceptibilities reported for Y. kristensenii and P. mirabilis.