The pineapple fruit when harvested generates a large amount of residual biomass; this biomass can be used to generate value-added products such as cellulose nanofibers. This study was focused on the isolation of CNF from pineapple leaves after oxidation pretreatment with 2,2,6,6-tetramethylpireridine-1-oxyl, followed by mechanical deconstruction of the fibers via combination of grinding and microfluidization process. One and two microfluidization passes were applied to bleached and unbleached fibers, respectively. The implications of these findings are that during the production process it is possible to reduce the amount of chemicals needed for bleaching and the energy involved in the mechanical microfluidization process. Such process yielded corresponding fibril lengths and widths in the range of 481–746 nm and 16–48 nm. The respective electrostatic charges, as measured by zeta potentials, were −41 mV and −31 mV. As expected, the CNF crystallinity was higher than that of the starting material, especially for the cellulose. However, the thermal stability was reduced, showing two degradative processes due to the chemical modification of the fibers. The CNF produced from pineapple leaves has a potential to be used like biomaterial in diverse applications while representing a viable alternative to producers, which face serious environmental and health challenges given the large volume of biomass that is otherwise left in the fields as waste. Graphic Abstract