An overview is given of our recent IR spectroscopic, optical, electrokinetic, and microscopic studies on polyelectrolyte multilayers (PEMs), built up using the consecutive layer-by-layer technique, their interactions, and application possibilities. The influence of pH on the deposition of PEMs consisting of commercial polyelectrolytes (PELs) like poly(ethyleneimine) and poly(acrylic acid) and the adopted surface morphology of isotropic PEMs consisting of flexible PELs are reported. PEMs of azo dye/PEL are included. Those are compared with anisotropic PEMs containing stiff PELs like charged α-helical polypeptides. Furhermore, examples concerning the swelling behavior of PEMs in the presence of solutions of different low molecular salt types and the hydration of dried PEMs by the relative humidity, respectively, are given. As potent application fields, their interaction with proteins regarding both prevention of bioadhesion and protein immobilization by electrostatic interaction forces are illustrated. Those studies led to principles for membrane surface modification in order to prevent biofouling and optimize their flux and separation performance.