The effect of different physico-chemical treatments used in the isolation process of aloe vera (AV) rind nanofibers on the tensile properties of the nanofiber films were studied to understand the root of the low strength values of these films. In the first stage of the investigation, ground AV rind was subjected to different chemical treatments before mechanical defibrillation. In the second stage, cuticle present in the AV rind was removed before subjecting them to chemical treatment. Nanofiber films were prepared using these defibrillated AV rind pulp fibers, and tensile properties were measured to examine the effect of chemical treatments and the raw material characteristics on the nanofibrillation process. The results showed that tensile values were not affected significantly with the chemical treatments; however, the thick cuticle coated on AV rind had a significant effect on the tensile strength and Young’s modulus of the nanofibre films. The maximum tensile strength and Young’s modulus (234.5 MPa and 12.6 GPa) of AV rind nanofibre films without cuticle were very high compared to the nanofibre films with the cuticle (tensile strength of about 110 MPa and Young’s modulus of 10 GPa). The study also found that mild chemical treatments were sufficient to isolate nanofibres from AV rind. The morphological analyses illustrated that the isolated AV rind nanofibers had a diameter size smaller than 20 nm and a high aspect ratio of approximately 1000 to 1500 or larger.