Controlling the bulk and surface properties of materials is a real challenge for bioengineers working in the fields of biomaterials, tissue engineering and biophysics. The layer-by-layer (LbL) deposition method, introduced 20 years ago, consists in the alternate adsorption of polyelectrolytes that self-organize on the material's surface, leading to the formation of polyelectrolyte multilayer (PEM) films. 1 Because of its simplicity and versatility, the procedure has led to considerable developments of biological applications within the past 5 years. In this review, we focus our attention on the design of PEM films as surface coatings for applications in the field of physical properties that have emerged as being key points in relation to biological processes. The numerous possibilities for adjusting the chemical, physical, and mechanical properties of PEM films have fostered studies on the influence of these parameters on cellular behaviors. Importantly, PEM have emerged as a powerful tool for the immobilization of biomolecules with preserved bioactivity.