A new technological approach to nanoparticle synthesis is using microorganisms, such as bacteria, which have the ability to synthesize nontoxic nanoparticles with high biocompatibility. In addition, bacteria have strict control over size, structure, shape. and dimension of produced nanoparticles. In the present work, Fe (III)-binding exopolysaccharide (Fe-EPS) nanoparticles were biosynthesized by Ralstonia pickettii sp. SK03, a bacterium isolated from a mineral spring. 16S rRNA gene sequencing and biochemical tests were done for identification of the isolated bacterium. For the first time, critical biological and physicochemical properties of this iron oxide nanoparticle were characterized using Fourier Transform Infrared (FTIR) Spectroscopy, Transmission Electron Microscopy (TEM), Vibrating Sample Magnetometer (VSM), Dynamic Light Scattering (DLS), Thermogravimetric analysis (TGA), Xray crystallography (XRD), Atomic absorption spectroscopy (AAS), and cell viability assays (M17 assay). The characterization results showed that Fe-EPS nanoparticles were composed of spherical ferrihydrite nanoparticles (with a size range of 1.2-2 nm), trapped in a polysaccharide matrix. The TGA analysis demonstrated that Fe-EPS nanoparticles contained similar to 25.2% polysaccharide. Therefore, this polysaccharide matrix showed a very low magnetic saturation value (0.25 emu/g) and a large negative charge of -93.8 mV. In addition, treatment of hepatocarcinoma cell line (Hep-G2) with 1-500 mu g/mL concentrations of Fe-EPS nanoparticles caused 40% increase in the cell viability, which indicated that the biosynthesized nanoparticles were nontoxic and biocompatible. (C) 2018 American Institute of Chemical Engineers