In this chapter, the authors review different biophysical studies performed with bacteriocins and reveal their importance in the process of developing an effective and selective anticancer peptides (ACP). Cancer remains a class of diseases with a significant detrimental impact on society. Even though many cancer patients survive, new therapies are essential for increasing survival rate and improving their life quality. ACPs have appealing features for industrial drug development. Bacteriocins are antimicrobial peptides produced by several types of bacteria, synthesized at the ribosome level. The increasing application of biophysical methodologies to the study of molecular events on bacteria and cancer cells has allowed the important development of knowledge about a cell’s biophysics, which without a doubt will positively influence the clinical application of bacteriocins in cancer treatment. Different studies show the importance of microscopy techniques for detailing bacteriocins’ mechanisms of action.