This paper discusses the critical role of thermal effect on the crystalline size, phase and energy band gap of nickel oxide nanoparticles synthesized utilizing the ASH SUPPORTED technique. These nanoparticles were baked from 300°C to 900°C. The phase of nickel oxide nanoparticles with isochronal annealing has been identified through structural characterization utilising X-ray diffraction of each sample. It was observed that nickel oxide nanoparticles have an average crystallographic size of about 26.2 nm. The pure phase of nickel oxide nanoparticles was obtained at 900°C, which was also supported by Raman spectra. Vibrational characteristics demonstrate the increase in intensity with temperature due to the improvement in the grain size. UV-Visible spectroscopy determines the direct variation of the band gap in the region of 2.14 eV - 3.37eV with varying crystallite size due to alteration in annealing temperature or may be due to surface & interface effect. It also exhibits excellent antibacterial activity against enteropathogenic bacteria Salmonella Sp, which shows zone of inhibition diameter of 10mm.