The bacterial nanocellulose has been used in a wide range of biomedical applications including carriers for drug delivery, blood vessels, artificial skin and wound dressing. The total of ten morphologically different bacterial strains were screened for their potential to produce bacterial nanocellulose (BNC). Among these isolates, Bacillus sp. strain SEE-3 exhibited potent ability to produce the bacterial nanocellulose. The crystallinity, particle size and morphology of the purified biosynthesized nanocellulose were characterized. The cellulose nanofibers possess a negatively charged surface of − 14.7 mV. The SEM images of the bacterial nanocellulose confirms the formation of fiber-shaped particles with diameters of 20.12‒47.36 nm. The TEM images show needle-shaped particles with diameters of 30‒40 nm and lengths of 560‒1400 nm. X-ray diffraction show that the obtained bacterial nanocellulose has crystallinity degree value of 79.58%. FTIR spectra revealed the characteristic bands of the cellulose crystalline structure. The thermogravimetric analysis revealed high thermal stability. Optimization of the bacterial nanocellulose production was achieved using Plackett–Burman and face centered central composite designs. Using the desirability function, the optimum conditions for maximum bacterial nanocellulose production was determined theoretically and verified experimentally. Maximum BNC production (20.31 g/L) by Bacillus sp. strain SEE-3 was obtained using medium volume; 100 mL/250 mL conical flask, inoculum size; 5%, v/v, citric acid; 1.5 g/L, yeast extract; 5 g/L, temperature; 37 °C, Na₂HPO₄; 3 g/L, an initial pH level of 5, Cantaloupe juice concentration of 81.27 percent and peptone 11.22 g/L.