There is a need for imaging and sensing instrumentation that can monitor transitions in a biofilm structure in order to better understand biofilm development and emergent properties such as anti-microbial resistance. Herein, we describe the design, manufacture, and use of a microfluidic flow cell to visualize the surface structure of bacterial biofilms with white-light interferometry (WLI). The novel imaging chip enabled the use of this non-disruptive imaging method for the capture of high resolution three-dimensional profile images of biofilm growth over time. The fine axial resolution (3 nm) and the wide field of view (>1 mm by 1 mm) enabled the detection of biofilm formation as early as 3 h after inoculation of the flow cell with a live bacterial culture (Pseudomonas fluorescens). WLI imaging facilitated the monitoring of the early stages of biofilm development and subtle variations in the structure of mature biofilms. Minimally-invasive imaging enabled the monitoring of biofilm structure with surface metrology metrics (e.g., surface roughness). The system was used to observe a transition in the biofilm structure that occurred in response to exposure to a common antiseptic. In the future, WLI and the biofilm imaging cell described herein may be used to test the effectiveness of biofilm-specific therapies to combat common diseases associated with biofilm formation such as cystic fibrosis and periodontitis.