Alkali-treated titanate layer with nanonetwork structures (TNS) is a promising surface for improving osseointegration capacity in implants. Nevertheless, there is a risk of device failure as a result of insufficient resistance to biofilm contamination. This study tested whether treatment using a handheld non-thermal plasma device could efficiently eliminate biofilm contamination without destroying the surface nanostructure while re-establishing a surface that promoted new bone generation. TNS specimens were treated by a piezoelectric direct discharge (PDD) plasma generator. The effect of decontamination was performed utilizing Staphylococcus aureus. The evaluation of initial cell attachment with adhesion images, alkaline phosphatase activity, extracellular matrix mineralization, and expression of genes related to osteogenesis was performed using rat bone marrow mesenchymal stem cells, and the bone response were evaluated in vivo using a rat femur model. Nanotopography and surface roughness did not significantly differ before and after plasma treatments. Cell and bone formation activity were improved by TNS plasma treatment. Furthermore, plasma treatment effectively eliminated biofilm contamination from the surface. These results suggested that this plasma treatment may be a promising approach for the treatment of nanomaterials immediately before implantation and a therapeutic strategy for peri-implantitis.