Bacterial cellulose (BC) has shown potential as a separation material. Herein, the performance of BC in pressure-driven separation is investigated as a function of incubation conditions and post-culture treatment. The pure water flux of never-dried BC (NDBC), was found to be 9 to 16 times higher than that for dried BC (DBC), in a pressure range of 0.25 to 2.5 bar. The difference in pressure response of NDBC and DBC was observed both in cross-flow filtration and capillary flow porometry experiments. DBC and NDBC were permeable to polymers with a hydrodynamic radius of ∼60 nm while protein retention was possible by introducing anionic surface charges on BC. The results of this work are expected to expand the development of BC-based filtration membranes, for instance towards the processing of biological fluids.