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Biofilms are microbial communities growing on surfaces, and are ubiquitous in nature, in bioreactors, and in human infection. Coupling between physical, chemical, and biological processes is known to regulate the development of biofilms; however, current experimental systems do not provide sufficient control of environmental conditions to enable detailed investigations of these complex interactions. We developed a novel planar flow cell that supports biofilm growth under complex two‐dimensional fluid flow conditions. This device provides precise control of flow conditions and can be used to create well‐defined physical and chemical gradients that significantly affect biofilm heterogeneity. Moreover, the top and bottom of the flow chamber are transparent, so biofilm growth and flow conditions are fully observable using non‐invasive confocal microscopy and high‐resolution video imaging. To demonstrate the …