Nanostructured materials have garnered significant attention due to their remarkable chemical and physical characteristics. Because of their exceptional qualities and potential uses in technology. Nanostructured transition metal oxides are one of the many nanomaterials that need particular attention. The goal of this study was to synthesize nickel oxide and barium oxide nanostructures by the reaction of Nano powders: nickel sulphate NiSO4, barium chloride BaCl3, and molten sodium hydroxide, potassium hydroxide (NaOH: KOH) and examine their physical and biological properties. According to the results of UV–Vis, the gap band of the nanostructure was calculated with a range of 3.6–3.27 eV. Scanning Electron Microscopy (SEM) pictures appear to create aggregated nanoparticles with particle size of 29 nm according to Transmission electron microscopy (TEM) with a spherical shape. This results proved that NiO: Ba nanostructure an efficient and stable. It was observed that the diameter of the inhibition zone bacteria include E. coli and Klebsiella spp. (21,18) mm while inhibition zone of bacteria S. aureus,S. epidermidisgram positive bacteria and yeast candida spp.(19,19 and 21)mm. As can be shown, the antibacterial activity of the (NiO: BaO), nanostructure increases with decreasing concentration and is more effective against E. coli gram-negative than it is against S. aureus and S. epedermidis gram-positive. This finding might be explained by the fact that, due to differences in their cell wall composition, gram-negative bacteria are more vulnerable to antibacterial medications than gram-positive ones. The synthesized NiO: Ba nanostructures had the potential for higher inhibition and killing of gram-negative than gram-positive bacteria.