Ultrathin films were prepared with cationic poly(allylamine hydrochloride) (PAH) and two anionic polysaccharides, ι- and λ-carrageenan, of similar chemical composition but different conformations using the layer-by-layer (LbL) technique. The study of aqueous solutions of carrageenans confirms that ι-carrageenan is at room temperature in helical conformation while λ-carrageenan is in random coil conformation. Characterization of the multilayers by ellipsometry, circular dichroism, and AFM revealed that ι-carrageenan keeps its helical conformation within the films while λ-carrageenan chains are in random coil conformation. Investigation of the mechanical properties of the films by performing nanoindentation experiments using force spectroscopy showed clear differences between the two films based on carrageenans of different conformations.