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Green synthesis of nanomaterials is advancing due to its ease of synthesis, inexpensiveness, nontoxicity and renewability. In the present study, an eco-friendly biogenic method was developed for the green synthesis of nickel oxide nanoparticles (NiONPs) using phytochemically rich Berberis balochistanica stem (BBS) extract. The BBS extract was rich in phenolics, flavonoids and berberine. These phytochemicals successfully reduced and stabilised the NiNO3 (green) into NiONPs (greenish-gray). BBS-NiONPs were confirmed by using UV-visible spectroscopy (peak at 305 nm), X-ray diffraction (size of 31.44 nm), Fourier transform infrared spectroscopy (identified -OH group and Ni-O formation), energy dispersive spectroscopy (showed specified elemental nature) and scanning electron microscopy (showed rhombohedral agglomerated shape). BBS-NiONPs were exposed to multiple in vitro bioactivities to ascertain their beneficial biological applications. They exhibited strong antioxidant activities: total antioxidant capacity (64.77%) and 2, 2-diphenyl-1-picrylhydrazyl (71.48%); and cytotoxic potential: Brine shrimp cytotoxicity assay with IC50 (10.40 µg/mL). BBS-NiONPs restricted the bacterial and fungal pathogenic growths at 1000, 500 and 100 µg/mL. Additionally, BBS-NiONPs showed stimulatory efficacy by enhancing seed germination rate and seedling growth at 31.25 and 62.5 µg/mL. In aggregate, BBS extract has a potent antioxidant activity which makes the green biosynthesis of NiONPs easy, economical and safe. The biochemical potential of BBS-NiONPs can be useful in various biomedical and agricultural fields.