In the present report, Nickel oxide nanoparticles (NiONPs) were synthesized using Rhamnus virgata (Roxb.) (Family: Rhamnaceae) as a potential stabilizing, reducing and chelating agent. The formation, morphology, structure and other physicochemical properties of resulting NiONPs were characterized by Ultra violet spectroscopy, X-ray diffraction (XRD), Fourier Transform Infrared analysis (FTIR), Scanning electron microscopy (SEM), Energy-dispersivespectroscopy (EDS), Transmission electron microscopy (TEM), Raman spectroscopy and dynamic light scattering (DLS). Detailed in vitro biological activities revealed significant therapeutic potential for NiONPs. The antimicrobial efficacy of biogenic NiONPs was demonstrated against five different gram positive and gram negative bacterial strains. Klebsiella pneumoniae and Pseudomonas aeruginosa (MIC: 125 μg/mL) were found to be the least susceptible and Bacillus subtilis (MIC: 31.25 μg/mL) was found to be the most susceptible strain to NiONPs. Biogenic NiONPs were reported to be highly potent against HepG2 cells (IC 50 : 29.68 μg/ml). Moderate antileishmanial activity against Leishmania tropica (KMH 23 ) promastigotes (IC 50 : 10.62 μg/ml) and amastigotes (IC 50 : 27.58 μg/ml) cultures are reported. The cytotoxic activity was studied using brine shrimps and their IC 50 value was recorded as 43.73 μg/ml. For toxicological assessment, NiONPs were found compatible towards human RBCs (IC 50 : > 200 μg/ml) and macrophages (IC 50 : > 200 μg/ml), deeming particles safe for various applications in nanomedicines. Moderate antioxidant activities: total antioxidant capacity (TAC) (51.43%), 2,2-diphenyl-1-picrylhydrazyl (DPPH) activity (70.36%) and total reducing power (TRP) (45%) are reported for NiONPs. In addition, protein kinase and alpha amylase inhibition assays were also performed. Our results concluded that Rhamnus virgata synthesized NiONPs could find important biomedical applications with low cytotoxicity to normal cells.