Chemically nickel oxide nanoparticles (NiONPs) involve the synthesis of toxic products, which restrict their biological applications. Hence, we developed a simple, eco-friendly, and cost-efficient green chemistry method for the fabrication of NiONPs using fresh leaf broth of Rhamnus triquetra (RT). The RT leaves broth was used as a strong reducing, capping, and stabilizing agent in the formation of RT-NiONPs. The color change in solution from brown to greenish black suggests the fabrication of RT-NiONPs which was further confirmed by absorption band at 333 nm. The synthesis and different physicochemical properties of RT-NiONPs were investigated using different analytical techniques such as UV-Vis (ultraviolet−visible spectroscopy), XRD (X-ray powder diffraction), FT-IR (Fourier-transform infrared spectroscopy), SEM (scanning electron microscopy), TEM (transmission electron microscopy), EDS (energy-dispersive X-ray spectroscopy), DLS (dynamic light scattering) and Raman. Further, RT-NiONPs were subjected to different in vitro biological activities and revealed distinctive biosafe and biocompatibility potentials using erythrocytes and macrophages. RT-NiONPs exhibited potential anticancer activity against liver cancer cell lines HUH7 (IC50: 11.3 µg/mL) and HepG2 (IC50: 20.73 µg/mL). Cytotoxicity potential was confirmed using Leishmanial parasites promastigotes (IC50: 27.32 µg/mL) and amastigotes (IC50: 37.4 µg/mL). RT-NiONPs are capable of rendering significant antimicrobial efficacy using various bacterial and fungal strains. NiONPs determined potent radical scavenging and moderate enzyme inhibition potencies. Overall, this study suggested that RT-NiONPs can be an attractive and eco-friendly candidate. In conclusion, current study showed potential in vitro biological activities and further necessitate different in vivo studies in various animal models to develop leads for new drugs to treat several chronic diseases.