In recent years, biosynthesis of nanoparticles has received considerable attention due to the growing need to develop clean and nontoxic chemicals, low-cost approaches, eco - friendly solvents and renewable materials. In the current study, the biosynthesis of TiO nanoparticles (TiONPs) was attained by a chemical and biosynthesized method by using the aqueous leaf extract of Trigonella foenum-graecum (TF-TiONP). TiO NPs were characterized by FTIR, UV, XRD, HR-TEM and HR-SEM methods. The X-ray diffraction displayed the existence of TF-TiONPs which is confirmed by the incidence of peaks at 25.28 corresponds to 101 anatase form. HR-SEM perceptions revealed that synthesized TiONPs were spherical in shape and the size of individual nanoparticles as well as a few aggregates was found to be 20-90 nm. The antimicrobial activities of biosynthesized nanoparticles (TF-TiONPs) were examined using Kirby-Bauer method. The TF-TiO nanoparticles showed significant antimicrobial activity against all the tested microorganisms.