Abstract Summary: Extraction processes of natural fibers can be performed by different procedures that include mechanical, chemical and biological methods. Each method presents different advantages or drawbacks according to the amount of fiber produced or the quality and properties of fiber bundles obtained. In this study, biological natural retting was satisfactorily used for obtaining banana fibers from plant bunches. However, the most important contribution of this work refers to the description of the hierarchical microstructural ordering present in banana fiber bundles in both bundle surface and inner region. The chemical composition of banana fiber bundles has been evaluated by FTIR spectroscopy. Through exposure time, the fiber bundle configuration presents small variations in composition. The main changes are related to hemicellulose and pectins as they conform the outer walls of the bundle. Hierarchical helicoidal ordering in the bundle surface as well as orientation on the longitudinal axis of the bundle were observed by optical microscopy (OM) and scanning electron microscopy (SEM) for 3–4 μm surface fibers and 10–15 μm inner elementary fibers, respectively. With increasing exposure time, fiber bundle walls lose integrity, as reflected in their mechanical behavior. magnified image