This work comprises the structural characterisation of Eucalyptus and Pinus radiata pulp fibres and their corresponding fibrillated materials, based on quantitative electron microscopy techniques. Compared to hardwood fibres, the softwood fibres have a relatively open structure of the fibre wall outer layers. The fibrillation of the fibres was performed mechanically and chemi-mechanically. In the chemi-mechanical process, the pulp fibres were subjected to a TEMPO-mediated oxidation to facilitate the homogenization. Films were made of the fibrillated materials to evaluate some structural properties. The thicknesses and roughnesses of the films were evaluated with standardized methods and with scanning electron microscopy (SEM), in backscattered electron imaging mode. Field-emission SEM (FE-SEM) and transmission electron microscopy (TEM) were performed to quantify the nanofibril morphology. In this study, we give additional and significant evidences about the suitability of electron microscopy techniques for quantification of nanofibril structures. In addition, we conclude that standard methods are not suitable for estimating the thickness of films having relatively rough surfaces. The results revealed significant differences with respect to the morphology of the fibrillated material. The differences are due to the starting raw material and to the procedure applied for the fibrillation.