This work systematically explores the bio-mineralization of calcium phosphate (CaP) and carbonate (CaCO3) within chitosan/iota-carrageenan multilayer films. Multilayer films of chitosan and iota-carrageenan (up to 128 coupled layers) were prepared on glass substrates by a layer by-layer dip-coating technique. Cryo-scanning electron microscopy revealed dense interfaces between the chitosan and iota-carrageenan layers with thicknesses in the range 250 and 350 nm in the hydrated state, accounting for the iridescent nature of multilayer films when wet. Immersion of the multilayered films in simulated body fluid or simulated seawater at 25 degrees C resulted in the mineralization of CaP and CaCO3, respectively, at the interfaces between the biopolymer layers and modified the iridescence of the films. Lamellar scattering features in small-angle neutron scattering measurements of the mineralized films provided evidence of the localized mineralization. Further evidence of this was found through the lack of change in the dynamic and static correlation lengths of the polymer networks within the bulk phase of the chitosan and iota-carrageenan layers. CaP mineralization occurred to a greater extent than CaCO3 mineralization within the films, evidenced by the higher lamellar density and greater rigidity of the CaP-mineralized films. Results provide valuable new insights into CaP and CaCO3 biomineralization in biopolymer networks.