A major by-product of biodiesel production is waste glycerol, which has numerous potential applications. In this study, we isolated a novel bacterium capable of producing cellulose from waste glycerol, and identified it as a novel strain (named NEDO-01) of Gluconacetobacter intermedius. Scanning electron microscopy revealed that the morphology of the pellicle produced by NEDO-01 was similar to that of cellulose produced by Gluconacetobacter hansenii ATCC23769. Furthermore, X-ray diffraction and solid-state nuclear magnetic resonance spectroscopic analyses suggested that cellulose produced by NEDO-01 had molecular and crystalline structures similar to those of cellulose produced by ATCC23769. After the optimization of cultivation conditions, NEDO-01 mediated the one-step production of nanofibrillated bacterial cellulose (NFBC) from waste glycerol in a medium supplemented with carboxymethyl cellulose. Transmission electron microscopic analysis revealed that the NFBC was composed of relatively uniform fibers with diameters of approximately 20 nm. NFBC was produced as uniform water suspensions, the yield of which was 3.4 g/L from cultivation in 7.5 L medium in a 10-L jar fermenter. The bioconversion of waste glycerol to NFBC, which has superior fluidity, moldability, and miscibility, has a wide variety of applications, including potential uses in the medical and materials engineering fields.