The effect of medium and ethanol on bacterial cellulose (BC) production by Gluconacetobacterxylinus (PTCC 1734) under agitating culture conditions was studied. The results showed that the yield and characteristics of BC are influenced by different components of the media. BC produced in Hestrin-Schramm (H) and Zhou (Z) media had the highest dry-weight yield, which increased by the addition of 1% (v/v) ethanol. According to Field Emission Scanning Electron Microscopy (FE-SEM) and Energy-Dispersive Spectroscopy (EDS) analyses, BC fibrils grown in Z medium were more intertwined than those grown in H and Y media. The addition of ethanol resulted in BC with a dense structure, and high O/C ratios. XRD and FT-IR measurements showed a higher crystallinity for the BC grown in H medium, while the BC grown in Z medium containing ethanol contained more surface carboxylate groups. This study shows that the BC properties can be altered by the culture medium and by the addition of ethanol.