In this paper, biocompatible calcium carbonate nanoparticles were produced from cuttlebone (Sepia Pharaonis) taken from Persian Gulf. Nanoparticles were synthesized by ball milling and emulsification methods and characterized by Field Emission Scanning Electron Microscopy (FESEM), Dynamic Light Scattering (DLS), Fourier transmission infrared spectroscopy (FTIR) and Brunauer–Emmett–Teller (BET) analysis techniques. Results showed that both methods (i.e. ball milling and surface surfactant) were able to produce calcium carbonate nanoparticles with an average diameter of 40 nm, and increasing the milling time could decrease nanoparticles diameter and increase their specific surface area. Comparison of these methods for biocompatible calcium carbonate nanoparticles production showed that the surfactant method has a potential for large-scale production of nanoparticles due to the simplicity of the process and low energy consumption. Also, ball milling method as a mechanical process strongly affects the specific surface area of nanoparticles. Performance of nanoparticles for wettability alteration of carbonate oil reservoirs was studied at different concentrations of nanofluids and temperatures. The results from measuring the contact angle between oil droplet and rock surface showed that increasing nanofluid concentration and temperature had a direct impact on increasing of the contact angle of oil droplet and wettability alteration from strongly oil-wet to the water-wet condition.