The aim of this study was to investigate the antibacterial effect and cytocompatibility of a nano-structured TiO2 film that contained Cl and had been coated onto commercially pure titanium. First, we prepared nano-structured TiO2 by anodization with hydrofluoric acid. Then, to confer an antibacterial effect, we performed a second anodization with NaCl solutions of different concentrations (0.5 M, 1 M, 2 M). The morphology, composition, and wettability of the surface were investigated by SEM, EDS, and a video contact angle measuring system. The antibacterial effect was evaluated by film adhesion method. And cytotoxicity was determined by the viability of MG-63 cells in a MTT assay. The SEM and EDS results showed that the TiO2 nano-structure containing Cl had successfully formed after the second anodization. The contact angle analysis showed that the anodized titanium had a hydrophilic character. The results of this in vitro investigation demonstrated that the TiO2 nano-structure film anodized in 1 M NaCl had an antibacterial effect and good cell compatibility.