Bacillus subtilis natto is the key microorganism for the industrial production of menaquinone-7. The fermentation of this bacterium in static culture is associated with biofilm formation. The objective of this study was to determine the effect of biofilm formation on menaquinone-7 production to develop a suitable bio-reactor for the production of menaquinone-7. In the static culture, menaquinone-7 biosynthesis showed a linear correlation with biofilm formation (R (2) = 0.67) and cell density (R (2) = 0.7). The amount of biofilm, cell density and menaquinone-7 formation were a function of nutrient and processing conditions. Glycerol, soy peptone, and yeast extract mixture and 40 °C were found to be the optimum nutrients and temperature for accelerating both biofilm and menaquinone-7 biosynthesis in static culture. However, glucose, mixture of soy peptone and yeast extract and 45 °C were found to be the optima for cell density. As compared to the static culture, the biofilm formation was significantly inhibited when a shaken fermentation was used. However, shaking caused only a small decrease on menaquinone-7 production. These results demonstrate that the biofilm formation is not essential for menaquinone-7 biosynthesis. This study underlines the feasibility of using large scale stirred fermentation process for menaquinone-7 production.