Biomass organization of Aspergillus niger biofilms and pellets stained with fluorescein isothiocyanate were analyzed by means of confocal laser scanning microscopy and detectable differences between both types of growth were found. Three-dimensional surface plot analysis of biofilm structure revealed interstitial voids and vertical growth compared with pellets. Growth was lower in biofilm and according to fluorescence profile obtained, biomass density increased at the surface (0-20 μm). However, a decrease in fluorescence intensity was observed through optical sections of pellets even though growth was significantly higher than biofilms. Cryo scanning electron microscopy also showed structural differences. While biofilms showed a spatially ordered mycelium and well structured hyphal channels, pellets were characterized by an entangled and notoriously compacted mycelium. These findings revealed common structural characteristics between A. niger biofilms and those found in other microbial biofilms. Thus, biofilm microstructure may represent a key determinant of biofilm growth and physiology of filamentous fungi. © 2009 Elsevier Ltd.