Metals that contain nanomaterials have the potential to be employed in controlling different kinds of infection, however, very limited information is known about their antibacterial properties. This study has been done to investigate the nanosynthesis titanium nanoparticles (TiNPs) using Streptococcus thermophilus and analyzing their biological actions as antibacterial. The bacterial isolates identified using universal primers 16S rRNA; then the 16S rRNA gene nucleotide sequences were aligned with the nucleotide sequences of strains obtained from the GeneBank through the software CLUSTAL X (version 1.82). Titanium nanoparticles were nanosynthesized by adding 0.025M titanium dioxide (TiO2) into cell-free supernatant for Streptococcus thermophilus. TiO2 was used as a precursor for nanobiosynthesis TiNPs. The formation of TiNPs was indicated by the color alteration of the solution from the light brown into dark brown indicates for the production of TiNPs. The Characterization of nanobiosynthesis was accomplished with UV-Visible (absorbance at 377nm), Scanning Electron Microscope, X-ray diffraction, Atomic Force Microscope, Energy-dispersive X-ray spectroscopy was used to distinguish the dimension, form (spherical) by SEM, dispersal (homogenous) and elemental analysis of nanoparticles. Biogenic TiNPs have displayed antibacterial and antibiofilm activity against both multidrugresistant Klebsiella pneumonia and Staphylococcus aureus. As an antibacterial activity, the TiNPs inhibited significantly K.pneumoniae (20 mm) with concentration (500 μg/ml), and S. aureus (16 mm) with the same concentration and increasing the concentration of TiNPs the inhibition zone increased. While as antibiofilm activity of TiNPs using the tube method, the tubes containing bacterial suspension K.pneumoniae and S.aureus with TiNPs, the results demonstrated that the biofilm formation was prevented and removed by the effect of TiNPs.