Bacterial cellulose (BC), a biopolymer, is synthesized by BC-producing bacteria. Almost all producing strains are classified in the family Acetobacteraceae. In this study, bacterial strain P285 was isolated from contaminated honey wine in a honey factory in northern Thailand. Based on 16S rRNA gene sequence identification, the strain P285 revealed 99.8% identity with Komagataeibacter mal-taceti LMG 1529 T. K. maltaceti P285 produced the maximum BC production at 20–30 °C and an initial media pH of 9.0. The highest BC production in modified mineral salt medium (MSM) was exhibited when glucose (16%, w/v) and yeast extract (3.2%, w/v) were applied as carbon and nitrogen sources, respectively. When sugarcane (8–16%, w/v) or honey (ratio of honey to water = 1: 4) supplemented with yeast extract was used, the BC production was greater. The characterization of BC synthesized by K. maltaceti P285 was undertaken using scanning electron microscopy (SEM) and Fourier transform infrared (FTIR) spectrometry. Meanwhile, X-ray diffraction results confirmed the presence of crystalline cellulose (2θ = 18.330, 21.390 and 22.640°). The maximum temperature of BC degradation was observed at 314 °C. Tensile properties analysis of hydrated and dried BC showed breaking strength of 1.49 and 0.66 MPa, respectively. These results demonstrated that K. maltaceti P285 has a high potential for BC production especially when grown in high initial media pH. Therefore, the strain would be suitable as an agent to make BC, the value-added product in the related factories.