To promote osteoblast adhesion and proliferation on (bio)material surfaces, biomimetic coatings resembling the natural extracellular matrix (ECM) are desirable. The glycosamino glycans (GAGs) chondroitin sulfate (CS) and heparin (HEP) are promising candidates for a biomimetic coating since they are two of the most prevalent noncollagenous biomolecules constituting the ECM. Coatings containing CS and HEP were prepared employing the "layer by layer" technique yielding polyelectrolyte multilayers (PEMs). Physicochemical and mechanical characterization of the coatings were performed by means of streaming potential measurements and colloidal force spectroscopy. The capability of the coatings to support cell adhesion, spreading, proliferation, and maintenance of an osteoblastic phenotype was assessed with SaOS osteosarcoma cells. We demonstrate that PEMs constructed from CS as the polyanion display a low Young's modulus correlated with poorly supported cell adhesion and proliferation. When the CS was adsorbed onto a stiffer polypeptide PEM basis, the Young's modulus increased, and the cell response was significantly improved. For HEP coatings an intermediate Young's modulus and moderate cell adhesion and spreading were observed. No significant changes in stiffness or cell response were detected when HEP was adsorbed onto the polypeptide film.