In this work, Musaceae isolated vascular bundles from rachis agro-industrial residues were evaluated as a potential source of cellulose microfibrils. For vascular bundle isolation, a biological retting was used. For cellulose microfibril isolation, two different alkaline treatments (sodium hydroxide or potassium hydroxide combined with bleaching and acid steps) were used in conjunction with a mechanical process. Cellulose microfibrils using both alkaline processes were successfully isolated, and the presence of non-cellulosic components, especially lignin and some hemicellulose as arabinose and galactose were reduced. In spite of an important amount of oxides being removed during the biological retting, XRF analysis revealed that calcium minerals were still present in the vascular bundles, and they can affect the cellulose microfibril isolation. AFM micrographs of isolated samples revealed cellulose microfibril bundles, and their presence can be associated with non-cellulosic components still present in the samples. Thermal analysis showed that when potassium hydroxide was used, a higher reduction of lignin was observed. Nevertheless, X-ray diffractions indicated no change in the crystallization pattern of cellulose I had occurred due to the isolation process used.