Digitonin, a steroidal saponin obtained from the foxglove plant (Digitalis purpurea), displays a wide spectrum of biological properties and is often used as a model in mechanistic investigations of the biological activity of saponins. In the present study, Langmuir monolayers of zwitterionic (DPPC, DMPE, POPC, POPE, DSPC, DSPE, and DPPE) and ionic (DPPS and DPPG) phospholipids were employed in order to better understand the effect of digitonin on the lipid organization. For this purpose, a combination of surface pressure relaxation, infrared reflection absorption spectroscopy (IRRAS), and fluorescence microscopy measurements was used. The observed increase in surface pressure (Π) suggests that digitonin can adsorb at the air/water interface, both bare and covered with the uncompressed phospholipid monolayers. However, the detailed analysis of IRRAS and fluorescence microscopy data shows that digitonin interacts with the lipid monolayers in a very selective way, and both the headgroup and the lipid tails affect this interaction. Nevertheless, it should be noted that in no case did digitonin cause any disruptive effects on the monolayers. The DPPE and DPPS monolayers get disordered by penetration with digitonin, despite an increase in surface pressure, leading to an unprecedented LC-LE transition. Interestingly, saponin could be easily squeezed out of these monolayers by mechanical compression.