Pigmentation of paper induced by fungi is one of the most complex phenomenons because it involves living organisms growing on a heterogeneous paper substrate. A novel approach to the study of interfaces of fungi and paper in black stains produced by pigmented Dematiaceous fungi with meristematic growth was undertaken applying surface metrology techniques: confocal laser scanning microscope (CLSM) white-light, confocal chromatic aberration profilometer (LCA) and scanning electron microscope in variable pressure (SEM-VP); fungal morphology was examined with transmitted light microscopy (TLM). The role of paper topography and surface morphology in fungi-induced biodeterioration was investigated elucidating some of the dynamic interactions of fungi with paper, spatial distribution of biological deposits, inclusions in paper matrix, and patterns of fungal growth on paper thus contributing to a better understanding of biodeterioration of paper-based cultural heritage. So far, to the authors' knowledge, there are no published reports on the investigation of interfaces of bio-stains and paper utilizing surface metrology techniques.