The combine use of ultrasonication and microbial fermentation represents a promising strategy for modifying the structural properties of cellulose derived from agricultural residues. This study investigated the synergistic effect of ultrasonication followed by fermentation with Aspergillus niger and Saccharomyces cerevisiae on the crystallinity index (CrI) and chemical composition of cellulose obtained from palm fruit fiber. Cellulose extracted from palm fruit fiber was subjected to ultrasonication and subsequently fermented using the selected microorganisms. Structural changes were analysed using X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR), while chemical composition was evaluated through one-way ANOVA (p < 0.05). The treatments significantly influenced cellulose crystallinity and chemical composition. Ultrasonication followed by A. niger fermentation resulted in the highest CrI (55.6%), whereas fermentation with S. cerevisiae produced the lowest CrI (39.4%). The results suggest that A. niger preferentially degrades amorphous cellulose regions, increasing relative crystallinity, while S. cerevisiae promotes greater structural disruption. Both microorganisms increased protein content and reduced dietary fiber levels. These findings demonstrate that combined physical and biological treatments can effectively modify cellulose structure, potentially improving its functionality for food and feed applications.