The taxonomy of fungal species, similar to that of many other microorganisms, suffers frequent revisions due to the discovery of new species and to the development and gathering of characterization data and morphological information. Morpho-taxonomy helps in the identification of many species. This work presents the macro, micro-morphological, and spectral mass analyses for phenotypic characterization of 13 species of Aspergillus section Nigri, showing that the characterization of spores (conidia) by scanning electron microscopy can be used as a tool to discriminate key morphological characteristics and separate closely related fungi. These results were corroborated by colony plates, stereomicroscopy, light microscopy, and spectral mass data.