A laboratory study was undertaken to extract nano-fibrillated cellulose (NFC) from the pseudostem (a waste from the fruit harvest) of two commercial banana cultivars (‘Grand Naine’ and ‘Poovan’) in Tamil Nadu using a novel approach: a one-step method with a bleaching agent and alkali-free acid hydrolysis coupled with ultrasonication. The acid hydrolysis was performed using nitric acid and acetic acid (1:10 ratio). The treatment was effective in the depolymerization and defibrillation of banana pseudostem fiber and in the formation of NFC, which was confirmed by several physico-chemical techniques. The average diameters of the nanofibrils were 6 to 8 nm and 4 to 6 nm for ‘Grand Naine’ and ‘Poovan’, respectively. The XRD analysis revealed an increased cellulose crystallinity of almost 20% in the NFC compared with the respective raw banana fibers. The Fourier transform infrared (FT-IR) spectroscopy and thermal gravimetric analysis (TGA) confirmed the absence of lignin, hemicelluloses, and pectin in the nano-fibrillated samples. The thermal analysis showed the increased thermal stability of the NFC.