Microalgae biofilms have been proposed as an alternative to suspended cultures in 10 commercial and biotechnological fields. However, little is known about their architecture which 11 may strongly impact biofilm behavior, bioprocess stability and productivity. In order to unravel the 12 architecture of microalgae biofilms, four species of commercial interest were cultivated in 13 microplates and characterized using a combination of confocal laser scanning microscopy and FTIR-14 spectroscopy. In all the species, the biofilm biovolume and thickness increased over time and 15 reached a plateau after 7 days, the final biomass reached was very different though. The roughness 16 decreased during maturation, reflecting cell division and voids filling. The extracellular polymeric 17 substances content of the matrix remained constant in some species and increased over time in some 18 others. Vertical profiles showed that young biofilms presented a maximum cell density at 20 µm 19 above the substratum co-localized with matrix components. In mature biofilms, the maximum 20 density of cells moved at a greater distance from the substratum (30-40 µm) whereas the maximum 21 coverage of matrix components remained in deeper layer. Carbohydrates and lipids were the main 22 macromolecules changing during biofilm maturation. Our results revealed that the architecture of 23 microalgae biofilms is species-specific. However, time is similarly affecting the structural and 24 biochemical parameters. 25