Carboxylated cellulose nanofibers were extracted from different biological species, including cotton, spruce wood, bamboo and bacterial cellulose, through a combined (2,2,6,6-tetramethyl-piperidine-1-yl)oxyl (TEMPO)-mediated oxidation and mechanical defibrillation treatment. The cross-section dimensions of isolated cellulose nanofibers in suspension were characterized by small-angle X-ray scattering, where the results were in agreement with the transmission electron microscopy measurement. For the spruce wood samples, the effects of three experimental variables in the combined TEMPO-oxidation and mechanical treatment on the dimensions and degree of oxidation of the resulting cellulose nanofibers were quantitatively investigated.