Altretamine (ALT) is a Food and Drug Administration (FDA) approved antineoplastic drug particularly used for ovarian cancer. This study examined, at a molecular level, the interactions of the drug with model membranes composed of phospholipids with different acyl chain lengths and head group charges at varied ALT concentrations based on temperature. For this purpose, spectroscopic studies of the liposomes in multilamellar vesicles form were conducted by Fourier transform-infrared spectroscopy (FTIR) and their calorimetric studies were carried out by differential scanning calorimetry (DSC) techniques. The results of the study showed that ALT clearly interacted with lipids and that these interactions were more significant in multilamellar vesicles made up of short chain phospholipids. Moreover, the results suggested that ALT settled into the tail group region, in particular the region that formed the hydrophobic part of lipids, and this effects the whole section of the membranes including glycerol backbones and head groups. This study is expected to contribute, on molecular level, to the studies on the knowledge of the mechanism in cancer, which is still very much a dangerous disease, and its related treatment.