Nanoparticles, fundamental to nanotechnology, are known for their distinct chemical, optical, and mechanical properties, which differ significantly from their bulk material counterparts. Due to increasing environmental toxicity concerns, this study focused on the eco-friendly biosynthesis of metallic nanoparticles (MNPs) using exo polysaccharides (EPS) extracted from endophytes of Triticum aestivum (wheat). The process involved dissolving 10mg of wheat seed polysaccharides in distilled water, followed by mixing with nickel nitrate to achieve a final concentration of 10 mM. The formation of nickel oxide nanoparticles (NiO-NPs) was indicated by a color change and confirmed through UV-visible spectroscopy. The green-synthesized MNPs were characterized using double-beam UV and FT-IR spectroscopy to identify the specific biomolecules involved in their formation, with X-ray diffraction (XRD)analysis determining their crystalline structure. The biological potential of these nanoparticles was assessed through various bioassays, including antioxidant activity evaluation using the DPPH radical scavenging method and hydrogen peroxide inhibition. Among all screened samples, NiO NPs showed the most significant antioxidant potential, with an EC50 value of 37.93 µg/mL, followed by EPS at 66.04 µg/mL. At 200 µg/mL, NiO NPs effectively scavenged theH2O2 free radical by 81.9%±0.24, followed by EPS at 76.54%±0.65. These results highlighted the effectiveness of NiO NPs, emphasizing their potential for sustainable applications in Nano medicine and Nano biotechnology.