Cardiolipins are essential for the integrity and the dynamics of the mitochondria membrane, where they exclusively exist in eukaryotes. Changes in cardiolipins membrane levels have been related to several cardiac health disorders. To evaluate cardiolipins impact on membrane properties a physico-chemical study was conducted using steady-state fluorescence anisotropy, dynamic light scattering and Nuclear Magnetic Resonance (1H and 31P NMR). Different binary and ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and a natural extract of bovine heart cardiolipin were used as models of mitochondrial membrane. The main transition temperatures, obtained by the first two techniques, revealed to be cardiolipins dependent. Cardiolipins also showed to act as a bidirectional regulator of membrane fluidity. 1H and 31P NMR results revealed that cardiolipins affects the conformation, mobility and structural order of the phospholipid molecules. According to 1H NMR results, cardiolipins disturbs the overall structure and packing order of membrane demonstrated with the decrease of the line broadening and shift of all resonances. The 31P NMR line shape analysis confirmed that, at distinct temperatures, different lipid phases coexist in the systems, and their type and quantitative distribution are cardiolipins dependent. In summary, cardiolipins presence/absence dramatically changes the membrane properties and has a major impact in the construction of a mitochondrial membrane model.