A novel pure and transparent bacterial nanocellulose (BNC) membrane was produced by in situ fermentation under static conditions using a defined minimal culture medium. BNC–Minimal membranes were characterized and compared with membranes produced in a usual complex medium, Mannitol. Morphological and physicochemical properties were investigated by scanning electron microscopy (SEM), BET-surface area, water holding capacity (WHC), X-ray diffractometry, light transmittance, refractive index, stress–strain measurements, and infrared spectroscopy (FTIR). Most importantly, the study was based on the use of a different culture medium and not on the addition of other components in the complex medium composition to make BNC-based composites more transparent. Our results revealed a high transparency of the BNC membranes produced in defined minimal medium, while the opacity of the usual BNC–Mannitol membranes was clearly noticed. BNC–Minimal membranes are pure cellulose and presented a different 3D microstructure, exhibiting porous surface on both sides. In addition, the BNC–Minimal membranes exhibit enhanced properties, like better elasticity, higher water holding capacity, greater surface area, higher crystallinity, and light transmittance. These results suggest that BNC–Minimal membranes are promising functional materials for several applications, including 3D platforms for better visualization of the cells, wound healing process that can be easily monitored without removing the dressing, optical devices. Moreover, their high elasticity is desirable for the design of blood vessels and skin substitutes that require expansive movements and flexibility.