As the substrate, tobacco waste extract (TWE) can produce bacterial cellulose (BC), a biobased material. However, nicotine inhibits BC production (adding 0.8 g/L nicotine to the HS medium had a negative effect on BC synthesis) and needs to be removed. In this study, BC production by Acetobacter oryzoeni MGC-N8819 was carried out in four dilutions (5 %, 10 %, 15 %, and 20 %) of TWE. 15 % TWE without nicotine removal resulting in a 3.27 g/L BC production. Considering the inhibitor effect of nicotine on BC synthesis. Pseudomonas sp. JY-Q/5∆, an efficient nicotine-degrading mutant strain without the ability of glucose consumption, was statically co-cultured with MGCN8819, and the BC production was increased to 4.61 g/L after 7 days of cultivation. To eliminate the limitation of insufficient oxygen supply, BC films were harvested on day 7 and cultured for an additional 5 days resulting in a 6.00 g/L final BC production. Remarkably, the co-culture of MGC-N8819 and JY-Q/5∆ improved BC properties in terms of fiber diameter (28 nm), mechanical properties (tensile strength to 67 MPa and elongation at break to 23 %), and thermal stability (the maximum decomposition temperature was 600 °C). This study suggests a valuable strategy for improving BC production using agricultural waste.