The excellent mechanical, tribological and biochemical properties of diamond coatings are promising for improving orthopedic or stomatology implants. A crucial prerequisite for such applications is an understanding and control of the biological response of the diamond coatings. This study concentrates on the correlation of diamond surface properties with osteoblast behavior. Nanocrystalline diamond (NCD) films (grain size up to 200 nm, surface roughness 20 nm) were deposited on silicon substrates of varying roughnesses (1, 270 and 500 nm) and treated by oxygen plasma to generate a hydrophilic surface. Atomic force microscopy was used for topographical characterization of the films. As a reference surface, tissue culture polystyrene (PS) was used. Scanning electron microscopy and immunofluorescence staining was used to visualize cell morphological features as a function of culture time. Metabolic activity, alkaline phosphatase activity, and calcium and phosphate deposition was also monitored. The results show an enhanced osteoblast adhesion as well as increased differentiation (raised alkaline phosphatase activity and mineral deposition) on NCD surfaces (most significantly on RMS 20 nm) compared to PS. This is attributed mainly to the specific surface topography as well as to the biocompatible properties of diamond. Hence the controlled (topographically structured) diamond coating of various substrates is promising for preparation of better implants, which offer faster colonization by specific cells as well as longer-term stability.