Objective: In recent decades, nanotechnology has been usedfor cancer diagnosis at early s tages, and also designing drugdelivery sys tems by targeting the nano-drug combination tocancer cells, and thus reducing the cytotoxicity to normal cells.Cobalt oxide is one of the mos t potent natural anticancer agentswhich has various applications in medicine, such as drug deliverysys tems, toxicity to cancer cells, and antibacterial properties.Cytotoxicity of cobalt oxide nanoparticles (Co۳O۴NPs)can be s tudied on various cell lines.Materials and Methods: In this s tudy, we firs t producedCo۳O۴ NPs using luminescence bacteria and then characterizedtheir properties and inves tigated their anticancer activityon colorectal cancer cells. The nanoparticles were characterizedby X-ray diffraction (XRD), transmission electron microscopy(TEM), scanning electron microscopy (SEM), and Fouriertransform-infra red spectroscopy (FTIR) analysis.Results: The XRD results illus trated the success in the synthesisof Co۳O۴NPs. The results of SEM and TEM indicatedthat the biologically synthesized cobalt oxide nanoparticles arespherical and cubic in shape with about ۷۰ nm diameter. Thesuccessful biosynthesis of cobalt oxide nanoparticles, was verifiedby the presence of amine groups as shown in FTIR analysis.The cytotoxicity s tudy exhibited a time and dose dependenteffect agains t CT۲۶ colorectal cancer cells as shown by MTTassay. The half maximal inhibitory concentration (IC۵۰) wasfound to be ۶۰ μg/ml after ۴۸ hours. The cytotoxic propertiesof Co۳O۴NPs were also s tudied on mouse embryonic fibroblas tcells (NIH۳T۳) as a normal cell line, where the results indicatedlower toxicity of the NPs compared to CT۲۶ cancerous cells.Conclusion: Based on our results, the biologically synthesizedCo۳O۴NPs were found to specifically decrease the viability ofCT۲۶ cancerous cells in vitro and thus they exhibit multifunctionalproperties.