Cellulose from different species of bamboo (Thyrsostachys siamesiGamble,Dendrocalamus sericeusMunro (DSM),Bambusa logispatha, andBambusasp.) was converted to cellulose nanocrystals (CNCs) by a chemical–mechanical method. First, bamboo fibers were pre-treated (removal of lignin and hemicellulose) to obtain cellulose. Next, the cellulose was hydrolyzed with sulfuric acid using ultrasonication to obtain CNCs. The diameters of CNCs are in the range of 11–375 nm. The CNCs from DSM showed the highest yield and crystallinity, which was chosen in the film fabrication. The plasticized cassava starch-based films with various amounts (0–0.6 g) of CNCs (from DSM) were prepared and characterized. As the number of CNCs in cassava starch-based films increased, water solubility and the water vapor permeability of CNCs decreased. In addition, the atomic force microscope of the nanocomposite films showed that CNC particles were dispersed uniformly on the surface of cassava starch-based film at 0.2 and 0.4 g content. However, the number of CNCs at 0.6 g resulted in more CNC agglomeration in cassava starch-based films. The 0.4 g CNC in cassava starch-based film was found to have the highest tensile strength (4.2 MPa). Cassava starch-incorporated CNCs from bamboo film can be applied as a biodegradable packaging material.