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• Polymorphism of formed NiO nanoparticles with size in 25-50 nm was observed. • High toxicity of nanoparticles was witnessed against MG-63 cancer cells. • NiO nanoparticles revealed high biocompatibility on HEK293 normal cells. • Nanoparticles showed bactericidal activity on various human pathogenic bacteria. The green synthesis of metal oxide nanoparticles is interestingly becoming familiar as the reason of its non-toxicity, economical, eco-friendly, and commerciality. The secondary metabolites and bioactive molecules in biomaterial extract play an important role of reducing, capping, stabilizing and chelating activities to form the nanoparticles as a substitute of hazardous chemical surfactants. Hence this present work concentrated on the biogenic synthesis of NiO nanoparticles using grains extract of Oryza longistaminata (red rice), and their toxicity assessment as well as biomedical applications. The synthesized nanoparticles were widely characterized by using several analytical and biomedical techniques. The reduction mechanism of Ni2+ ions using major active phytocompound in red rice grains extract was stated. UV–visible spectroscopic analysis revealed the formation of NiO nanoparticles by emerging the sharp surface Plasmon resonance band at 326 nm. The absorption peaks of red rice grains extract at 206, 260, 330 and 410 nm stated the presence of phytocompounds in them. X-ray diffraction pattern explicated the high crystallinity of NiO nanoparticles and their average crystallite size was found to be at 36 nm. The diffraction pattern of red rice grains revealed the amorphous characteristic. Fourier transform infrared analysis displayed the different vibrational functional groups of nanoparticles and red rice grains. Dynamic light scattering analysis showed the particle size distribution was in between 30-65 nm. High-resolution transmission electron microscopic analysis showed the hexagonal, rectangular, oval and spherical shaped polymorphism of NiO nanoparticles and their size was in 25-50 nm. Energy dispersive X-ray spectrum and mapping analyses validated the formation of NiO nanoparticles by the major existence of Ni and O elements. The high toxicity of nanoparticles towards MG-63 osteosarcoma cancer cells was revealed at 94.69% in 100 μg/mL concentration. The nanoparticles showed no severe toxicity against HEK293 normal cells and red blood cells till higher concentration. The biomedical analyses such as antioxidant, anti-inflammatory and anti-diabetic activities exposed the potential bio-drug activity of NiO nanoparticles with maximum activity of 86.22, 84.23 and 85.18% in the high concentration of 80 μg/mL. The antibacterial activity divulged the good bactericidal drug nature of NiO nanoparticles against both Gram-positive (S. aureus and B. Subtilis) and Gram-negative (S. typhi and K. pneumonia) bacteria with low MBC/MIC ratio. As per the assessment of results, the biosynthesized NiO nanoparticles have non-carcinogenic, high biocompatible and potent nature and it suggests that they can be used as therapeutic candidates in biomedical, clinical and pharmaceutical fields for treating various diseases. [Display omitted] [ABSTRACT FROM AUTHOR]