Bacteria are almost always found in ecological niches as matrix-encased, surface-associated, multi-species communities known as biofilms. It is well established that soluble chemical signals produced by the bacteria influence the organization and structure of the biofilm; therefore, there is significant interest in understanding how different chemical signals are coordinately utilized for community development. Conventional methods for investigating biofilm formation such as macro-scale flow cells are low-throughput, require large volumes, and do not allow spatial and temporal control of biofilm community formation. Here, we describe the development of a PDMS-based two-layer microfluidic flow cell (μFC) device for investigating bacterial biofilm formation and organization in response to different concentrations of soluble signals. The μFC device contains eight separate microchambers for cultivating biofilms exposed to eight different concentrations of signals through a single diffusive mixing-based concentration gradient generator. The presence of pneumatic valves and a separate cell seeding port that is independent from gradient-mixing channels offers complete isolation of the biofilm microchamber from the gradient mixer, and also performs well under continuous, batch or semi-batch conditions. We demonstrate the utility of the μFC by studying the effect of different concentrations of indole-like biofilm signals (7-hydroxyindole and isatin), either individually or in combination, on biofilm development of pathogenic E. coli. This model can be used for developing a fundamental understanding of events leading to bacterial attachment to surfaces that are important in infections and chemicals that influence the biofilm formation or inhibition.