Significance This report describes an opportunity for the bacterial cellulose nanofiber (BCNF) membrane, which has recently been attracting much attention due to its sustainability and high thermal stability, as a “single-layer,” good alternative membrane to the existing chemical-derived one in batteries, and thus allows us to find possible solutions to the environmental and safety issues. We achieved highest BCNF yield ever reported so far and fabricated a cylindrical lithium ion battery (LIB) by roll-to-roll process to realize remarkable cycle stability with 80% capacity retention even after 1,000 cycles, which is comparable to those of commercialized batteries. Finally, we introduced a Lewis base to improve thermal safety of the battery to a level comparable to that of a commercial ceramic-coated separator.