Pseudomonas aeruginosa is a particularly problematic opportunistic pathogen due to its capacity to form recalcitrant biofilm structures, while cohabiting with other harmful/pathogenic species and harboring the capability to release toxins that cause tissue necrosis. Although it is now recognized that the majority of biofilm infections are polymicrobial, little is known about the complex interactions that occur within polymicrobial communities and few tools exist for studying these interactions. In this study, we have designed a microfluidic model that mimics the relevant physiological properties of wound microenvironment, while incorporating materials present in the human extracellular matrix/wound environment. Using microfluidics combined with imaging techniques, we have validated the robustness of our model comparing traditional GFP-tagging to new fluorescent staining techniques to visualize/resolve individual species within a polymicrobial habitat. We have also demonstrated that chemotactic stimuli may be incorporated into our model through specialized ports in our chamber. Our system is specifically designed for use with high resolution imaging techniques, allowing for data collection throughout the life of the biofilm and in real-time. Ultimately, this model can be used to investigate the spatio-temporal mechanobiological structures of the wound environment, and the response of the bacteria to the drug transport which will significantly contribute to our understanding of the development and progression of polymicrobial biofilm infections. Biotechnol. Bioeng. 2015;112: 2351-2359. (c) 2015 Wiley Periodicals, Inc.