Cellulose nanofibers (CNF) were isolated from raw cotton fibers via 2, 2, 6, 6-tetramethylpiperidine-1-oxyl (TEMPO) oxidation process. The isolated CNF were morphologically characterized using Field Emission Scanning Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM). Entangled fibrous morphology with diameter in nano regime (15–20 nm) has been observed for the isolated nanofibers. X-ray diffraction (XRD) analysis confirms the high crystallinity of the isolated CNF. Further, the isolated CNF was used to reinforce natural rubber (NR) latex films. The crystallographic, morphological, and spectroscopic analysis of the NR/CNF nanocomposites was carried out. A strong interaction between the CNF and NR matrix has been identified and which reflects in the thermal stability and swelling behavior of NR/CNF nanocomposite films in toluene. The uniform dispersion and tangling effect of CNF along with strong NR-CNF interaction restrict the uptake of solvent through NR matrix.