The surface engineering of biomaterials is crucial for their successful (bio)integration by the body, i.e. the colonization by the tissue-specific cell, and the prevention of fibrosis and/or bacterial colonization. Performed at room temperature in an aqueous medium, the layer-by-layer (LbL) coating method is based on the alternating deposition of macromolecules. Versatile and simple, this method allows the functionalization of surfaces with proteins, which play a crucial role in several biological mechanisms. Possessing intrinsic properties (cell adhesion, antibacterial, degradable, etc.), protein-based LbL films represent a powerful tool to control bacterial and mammalian cell fate. In this article, after a general introduction to the LbL technique, we will focus on protein-based LbL films addressing different biomedical issues/domains, such as bacterial infection, blood contacting surfaces, mammalian cell adhesion, drug and gene delivery, and bone and neural tissue engineering. We do not consider biosensing applications or electrochemical aspects using specific proteins such as enzymes