This study describes the effect of multilayered DNA coatings on (i) the formation of mineralized depositions from simulated body fluids (SBF); and (ii) osteoblast-like cell behavior with and without pretreatment in SBF. DNA coatings were generated using electrostatic self-assembly, with poly-d-lysine or poly(allylamine hydrochloride) as cationic polyelectrolytes, on titanium substrates. Coated substrates and non-coated controls were immersed in SBF with various compositions. The deposition of calcium phosphate was enhanced on multilayered DNA coatings as compared with non-coated controls, and was dependent on the type of cationic polyelectrolyte used in the build-up of the DNA coatings. Further analysis showed that the depositions consisted of carbonated apatite. Non-pretreated DNA coatings were found to have no effect on osteoblast-like cell behavior compared with titanium controls. On the other hand, SBF-pretreatment of DNA coatings affected the differentiation of osteoblast-like cells through an increased deposition of osteocalcin. The results of this study are indicative of the bone-bonding capacities of DNA coatings. Nevertheless, future animal experiments are required to provide conclusive evidence for the bioactivity of DNA coatings.