We report the intrinsic biological properties of nickel oxide nanoparticle (NiO NPs) encapsulated Nardostachys jatamansi DC-based polymethyl methacrylate (PMMA) via free radical polymerization technique. A simple and cost effective NiO NPs was adapted to synthesize via sol-gel/co-precipitation method to afford crystalline structures of NPs and it was confirmed by X-ray diffraction (XRD) patterns (2θ at 38.78 [111] and 62.66 [220]). The results demonstrated that the sparingly loaded NiO NPs onto composites exhibited the lowest crystallinity, which led to better dispersion into the polymer matrix and also observed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). ATR-IR enunciate the functional groups of polymer nano-biocomposites attributed at 1730 cm −1 ( C=O), 1446 cm −1 ( CH 3 -asymmetric deformation), 1162 cm −1 ( C-N), and 822 cm −1 ( C-H), which were found to be good agreement for the formation of polymer composites and further confirmed their structural identification by NMR, fluorescence spectroscopy and UV-vis spectroscopy. This hybrid nano-biocomposite has been utilized for antimicrobial studies using a gram positive and gram-negative bacteria and showed better results on minimum bactericidal concentration (MBC) assay against Micrococcus luteus, Staphylococcus epidermidis, Yersinia enterocolitica, and Klebsiella pneumoniae. In addition, cell morphology and proliferation were monitored by fluorescence microscopy. The MTT assay results showed that the nano-biocomposites possessed significantly low cytotoxicity against human keratinocyte cell lines (HaCaT).