Bioactive peptides have emerged as promising therapeutic agents with antimicrobial, antifungal, antiparasitic, and, recently, antitumoral properties with a mechanism of action based on membrane destabilization and cell death, often involving a conformational change in the peptide. This biophysical study aims to provide preliminary insights into the membrane-level antitumoral mode of action of crotalicidin, a cationic host defense peptide from rattlesnake venom, toward breast cancer cell lines. The lipid composition of breast cancer cell lines was obtained after lipid extraction and quantification to prepare representative cell membrane models. Membrane–peptide interaction studies were performed using differential scanning calorimetry and Fourier-transform infrared spectroscopy. The outcome evidences the potential antitumoral activity and selectivity of crotalicidin toward breast cancer cell lines and suggests a mechanism initiated by the electrostatic interaction of the peptide with the lipid bilayer surface and posterior conformation change with membrane intercalation between the acyl chains in negatively charged lipid systems. This research provides valuable information that clears up the antitumoral mode of action of crotalicidin.