Cobalt is essential to the metabolism of all animals due to its key role in cobalamin, also known as vitamin B12, the primary biological reservoir of cobalt as an ultra-trace element. Current cancer treatment strategies, including chemotherapy and radiotherapy, have been seriously restricted by their side effects and low efficiency for a long time, which urges us to develop new technologies for more effective and much safer anticancer therapies. Novel nanotechnologies, based on different kinds of functional nanomaterials, have been proved to act as effective and promising strategies for anticancer, microbiological, antioxidant, antidiabetic, anti-inflammatory and other treatments. Based on the important biological roles of cobalt, cobalt oxide nanoparticles (NPs) have been widely developed for their attractive biomedical applications. Co3O4-Nps were synthesized by using leaf extract of Boerhavia diffusa and cobalt nitrate hexahydrate as a source of cobalt. The synthesized NPs were characterized by different techniques such as Ultra-violet spectroscopy (UV), Fourier transform infrared spectroscopy (FTIR), Fluorescence spectroscopy (FL), X-ray diffraction (XRD), Dynamic light scattering (DLS) analysis, scanning electron microscopy (SEM) with EDS and TEM. Electrical property analyzed by cyclic voltammetry (CV). The synthesized Co3O4-Nps were evaluated in vitro petri plate disc diffusion methods for antibacterial and antifungal activities, anti-oxidant, anti-diabetic, anti-inflammatory studies were done by enzymes denaturation methods, anticancer activity of Co3O4-Nps have examined by HepG2 cell line.