A strain of acetic acid bacteria, Komagataeibacter xylinus B-12068, was studied as a source for bacterial cellulose (BC) production. The effects of cultivation conditions (carbon sources, temperature, and pH) on BC production and properties were studied in surface and submerged cultures. Glucose was found to be the best substrate for BC production among the sugars tested; ethanol concentration of 3% (w/v) enhanced the productivity of BC. Optimization of medium and cultivation conditions ensures a high production of BC on glucose and glycerol, up to 2.4 and 3.3 g/L/day, respectively. C/N elemental analysis, emission spectrometry, SEM, DTA, and X-ray were used to investigate the structure and physical and mechanical properties of the BC produced under different conditions. MTT assay and SEM showed that native cellulose membrane did not cause cytotoxicity upon direct contact with NIH 3T3 mouse fibroblast cells and was highly biocompatible.