Bacterial nanocellulose (BNC) membranes are emerging as a novel 3D platform for studies of cell-cell interaction and cell migration. However, the opacity of the membranes partially obscures visualization and prevents live observation of cultures. Our proposal was to develop a BNC membrane with enhanced optical transparency. BNC membranes synthesis by Gluconacetobacter hansenii ATCC 23769 was investigated using three media formulations: Mannitol, Hestrin–Schramm and a modified minimal medium. Measurements of morphological and mechanical properties were investigated by scanning electron microscopy (SEM), infrared spectroscopy (FTIR) and stress–strain measurements. Our results revealed that the BNC produced by our minimal media were more optically transparent than the other membranes and can be used as a platform for 3D studies in tissue engineering in which cells do not have to be sacrificed, and cultures can be visualized throughout the experimental time course.