The need for energy-storing technologies with lower environmental impact than Li-ion batteries but similar power metrics has revived research in Zn-based battery chemistries. The application of bio-based materials as a replacement for current components can additionally contribute to an improved sustainability of Zn battery systems. For that reason, bacterial cellulose (BC) was investigated as separator material in Ni-Zn batteries. Following the biotechnological production of BC, the biopolymer was purified, and differently shaped separators were generated while surveying the alterations of its crystalline structure via X-ray diffraction measurements during the whole manufacturing process. A decrease in crystallinity and a partial change of the BC crystal allomorph type Iα to II was determined upon soaking in electrolyte. Electrolyte uptake was found to be accompanied by dimensional shrinkage and swelling, which was associated with partial decrystallization and hydration of the amorphous content. The separator selectivity for hydroxide and zincate ions was higher for BC-based separators compared to commercial glass-fiber (GF) or polyolefin separators as estimated from the obtained diffusion coefficients. Electrochemical cycling showed good C-rate capability of cells based on BC and GF separators, whereas cell aging was pronounced in both cases due to Zn migration and anode passivation. Lower electrolyte retention was concluded as major reason for faster capacity fading due to zincate supersaturation within the BC separator. However, combining a dense BC separator with low zincate permeability with a porous one as electrolyte reservoir reduced ZnO accumulation within the separator and improved cycling stability, hence showing potentials for separator adjustment.