The shell microstructure of freshwater bivalve Anodonta cygnea was observed from the ventral margin toward the intrapallial regions by scanning electronic microscopy during the spring/summer period. The three main structures—the periostracum, prismatic, and nacreous layers—were visualized in a sequential path from the ventral margin toward the intrapallial regions. Although with always the same calcium carbonate polymorph (aragonite), A. cygnea presented composites of aragonite crystals embedded within an organic matrix displaying varied morphologies and structural arrangements. The prisms of the prismatic shell layer are composed of single-crystal fibers radiating from spheruliths, whereas in the nacreous layer the structure is similar to that of a brick wall, with tablets in each layer offset with respect to those in the layers above and below them. From the shell ventral margin, the last nacreous lamina formed exhibit crystals with rounded forms, followed by rhombic and hexagonal shapes toward the interior with irregular microstructure. There are gradual intermediate forms between these distinct shapes. This study offers a description of the inner calcareous mineralized layers of A. cygnea and will be useful for future comparative studies in several research areas such as phylogenetic evolution, ecotoxicology, and the influence of environmental and physicochemical factors on the shell formation process in freshwater bivalves.