The prismatic layer of Pinna (Mollusca) is one of the most studied models for the understanding of the biomineralization mechanisms, but our knowledge of the organic components of this layer is limited to the proteins of the soluble organic matrices. The interplay of the mineral and organic matrices is studied using scanning electron and atomic force microscopy, infra-red spectrometry, thermogravimetric analyses, aminoacids analyses, thin layer chromatography (TLC), X-ray fluorescence, X-ray Absorption near Edge Structure (XANES) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Proteins, polysaccharides and lipids are detected within the prisms and their envelopes. The role of the technical choices to study calcareous biominerals is evidenced, showing that a single analysis is not enough to decipher complex biominerals.