AbstractBacterial cellulose (BC) synthesized by Gluconacetobacter sucrofermentans has a high degree of crystallinity, durability, great resistance, and biocompatibility. This study evaluates the effect that carbon and nitrogen sources, present in HS (Hestrin–Schramm) culture medium, have on BC from G. sucrofermentans CECT 7291 used to restore damaged documents. The initial pH of the medium and the temperature were set for all assays, which were conducted in static conditions. The cellulose layers were obtained at four time points. The growth media were characterized at each time point (pH and carbon source consumption). Cellulose layers were washed, pressed, dried, and characterized by determining their pH, dry weight, and optical and mechanical properties. The best combination of carbon and nitrogen sources proved to be fructose plus yeast extract–corn steep liquor, with or without ethanol, which provided a good balance between BC production and carbon source consumption, and generated a resistant and homogeneous cellulose layer.