Vitamin B2 (riboflavin) is the primary metabolite produced in industrial quantities by genetically modified strains of Bacillus subtilis. Different media were tested in shake cultures to obtain the one with the highest productivity of the selected production organism. To improve the understanding of B. subtilis growth and riboflavin production, we have developed a microfluidic reactor for biofilm cultivation. In this reactor, oxygen transfer tests were carried out to confirm that the reactor is suitable for the cultivation of aerobic microorganisms. Biofilms of B. subtilis were grown at different flow rates and different channel geometries. We found that a sufficiently fast medium flow rate of at least 1 μl/min was required for the formation of stable biofilms. Biofilms were grown in the microfluidic reactor in different media and with different amounts of oxygen. We found that the amount of oxygen available has a strong influence on biofilm formation and development, as well as on the morphological characteristics of the biofilm. In the developed microreactor system, due to the short retention times, the bacterium did not produce sufficient amounts of riboflavin for it to be monitored in an integrated analytical system.