The present work studies anodic TiO2 nanotube (TNT) layers and their surface modifications for enhancing the cell behavior of human gingival fibroblast cells (hGFs) with the contribution of bio-AFM (Atomic Force Microscopy) method. TNT layers, prepared via electrochemical anodization of Ti, with an average tube diameter of 15, 30, and 100 nm, were used as primary substrates for the study. Flat Ti foils were used as reference substrates. Part of the substrates was coated by ultrathin TiO2 coatings (≈ 0.3 nm thin) using Atomic Layer Deposition (ALD). The cell growth and adhesion of hGFs on the TNT layers and Ti foils were compared between ALD coated and uncoated ones. The supplemental coatings altered the surface chemistry of the TNT layers, particularly shading the fluorine and carbon impurities within anodic TiO2, while preserving the original structure and morphology. The presented approach of very mild surface modification remarkably effects the material's biocompatibility and possess great prospect as implant materials. For the first time, the TNT/cell interface was investigated using bio-AFM in terms of Young's modulus, stiffness, cell adhesive force and roughness. Improved biocompatibility was studied in terms of increased cell viability, density, cell cytoskeleton orientation and overall stiffness of the hGFs.