Layer-by-layer films based on chitosan and hyaluronic acid were produced by dip- and spin-coating techniques onto glass, 316L stainless steel and titanium. These natural polymers were modified with catechol groups, in order to build coatings with improved adhesive properties. Polymeric coatings were exclusively composed by both modified polymers whereas the multifunctional coatings combined an inorganic phase of bioactive glass nanoparticles with the polymeric layers to confer bioactivity. Ultraviolet-visible spectroscopy demonstrated that both polymers were successfully synthesized. Fourier transform infrared imaging was used as an innovative way to analyze the layer interdiffusion in these coatings. Their morphology was analyzed by scanning electron microscopy and atomic force microscopy, and their wettability was evaluated by water contact angle measurements. Major differences were found in the structure and surface properties of the coatings assembled either by dip- or spin-coating. The spin-coated films onto glass were smoother, with a more homogeneous structure and lower interdiffusion of polyelectrolytes layers, when compared with the dip-coated ones. Furthermore, it was concluded that the intrinsic surface roughness of stainless steel and titanium substrates had great influence on the surface morphology and wettability of the coatings obtained from both layer-by-layer methodologies.