Abstract: Acetobacterium woodii, an anaerobic acetogenic bacterium was used in this study to investigate the biotransformation of guaiacol in batch culture via O-demethylation reaction. The culture was grown anaerobically using 250 mL conical flask containing guaiacol in the presence or the absence of fructose. Results showed that limiting fructose concentration for the highest cell growth was 2.0 g L-1. It is also found that 94% of guaiacol was transformed to catechol (0.56 g L-1) when 2.0 g L-1 of fructose and 0.6 g L-1 of guaiacol were used as carbon source for growth. In cultures containing 2 g L-1 fructose (with addition of 0.6 g L-1 guaiacol after 20 h incubation), the catechol production was 0.5 g L-1 (82% transformation), while in cultures containing only guaiacol, the catechol production was 0.4 g L-1 (62% transformation). In the experiment using harvested cells, the highest catechol production was 0.47 g L-1 (79% transformation). The highest transformation was obtained using non-growing harvested cells from cultures containing 2 g L-1 fructose with 0.6 g L-1 guaiacol addition suggesting that this is the best combination of carbon source for high production of catechol. It is also suggested that high cell concentration in culture containing limited concentration of fructose will resulted in high concentration of catechol believed to be due of stimulation of the enzyme for O-demethylation reaction.