Fructooligosaccharides from chicory (FOSC) are functional prebiotic foods recognized to exert several well-being effects in human health and animal production, as decreasing blood lipids, modulating the gut immune system, enhancing mineral bioavailability, and inhibiting microbial growth, among others. Mechanisms of actions directly on cell metabolism and structure are however little known. In this sense this work was targeted to investigate the interaction of FOSC with biomimetic membranes (liposomes and supported bilayer membrane; s-BLM) through cyclic voltammetry, impedance spectroscopy, spectrofluorimetry, and microscopy. FOSC was able to disrupt the membrane structure of liposomes and s-BLM from the onset of molecular pores induced on it. The mechanism of interaction of fructans with biomimetic membranes suggests hydrogen bonding between the polyhydroxylated structure of the oligosaccharides and the negative polar group of L-α-phosphatidylcholine (PC) present in both liposomes and s-BLM.