Comprehending how raw materials, pretreatments, and treatments affect the properties of cellulose nanofibrils (CNFs) is crucial for their use. This study aims to understand the effect that alkaline treatment has on the characteristics of pulps, the mechanical processes of CNF production, and the characteristics of CNFs from Pinus radiata and Eucalyptus nitens pulps with native and oxidized lignin. For this purpose, Pinus radiata and Eucalyptus nitens pulps with different lignin contents produced by oxidative pretreatment were used as raw material. From these, pulps with different hemicellulose and lignin contents were produced by alkaline treatment. Furthermore, CNFs were prepared by refining and homogenization processes. The chemical composition analysis of pulps containing native lignin, before and after alkaline treatment, revealed distinct behaviors of hemicellulose. In Pinus radiata pulp, hemicellulose remained insoluble under alkaline conditions. In contrast, the hemicellulose from Eucalyptus nitens pulp was partially soluble. Morphological characteristics of CNFs revealed that removing 41.8% of total hemicellulose by alkaline treatment promoted the mechanical fibrillation of Eucalyptus nitens pulp with native lignin, decreasing the average width by 30%. Furthermore, a very low lignin content in this species (similar to 1.3%) hindered the mechanical fibrillation of fibers. Finally, the dielectric spectra of CNFs showed that the alkaline treatment increased the activation energies of the relaxations associated with the molecular motions of the hemicellulose and lignin groups, evidencing changes in their structures. These changes were related to the deacetylation of hemicellulose, and the deprotonation of hydroxy groups and the formation of carboxyl groups in lignin.