Bacterial cellulose (BC) is a nanostructured material mainly produced by Gluconacetobacter. The excellent physicochemical and mechanical properties of BC have made it become regarded as a kind of highly functional biopolymer in the application fields of bio-medicine, cosmetics and food industry. However, application of bacterial cellulose faced one of the main big problems in industry, viz., low productivity. Herein, this work was undertaken with a view to enhance the BC production using Komagataeibacter xylinus SB3.1 under static condition through study the effective culture parameters that played a vital role in nanofiber production. Various parameters were investigated including, carbon and nitrogen sources, inoculum size, pH, temperature, and incubation time. Chemical and physical characterizations were investigated using state-of-art tools e.g. FTIR, XRD and SEM. Results revealed the successful production of BC nanofibers through using Komagataeibacter xylinus SB3.1 under static condition as obviously indicated from SEM image. FTIR and XRD affirmed both chemical structure and crystallographic nature of cellulose I of the produced nanofibers. In the seek of improvement, the maximum yield production of BC reached 6.54 g/l at optimum conditions including the use of mannitol and yeast extract as a sole carbon and nitrogen source during incubation period for 8 days at 30 ◦C with 8% inoculum size. These findings emphasize the potential use of a new locally isolated Komagataeibacter xylinus SB3. in production of BC nanofibers as cellulose type I with enhanced crystallinity index up to 82% occurred at optimum conditions which reveals to the increase in the fiber production 3.3 folded times than others with unoptimized condition under the same stationary conditions of growth. This also opens up the window for utilizing the domestic isolated Acetobacter bacteria in industrial manufacturing with potential features.