We describe the use of optical coherence tomography (OCT) for high-resolution, real-time imaging of three-dimensional structure and development of a Pseudomonas aeruginosa biofilm in a standard capillary flow-cell model. As the penetration depth of OCT can reach several millimeters in scattering samples, we are able to observe complete biofilm development on all surfaces of a 1 mm×1 mm flow-cell. We find that biofilm growing at the bottom of the tube has more structural features including voids, outward projections, and microcolonies while the biofilm growing on the top of the tube is relatively flat and contains less structural features. Volume-rendered reconstructions of cross-sectional OCT images also reveal three-dimensional structural information. These three-dimensional OCT images are visually similar to biofilm images obtained with confocal laser scanning microscopy, but are obtained at greater depths. Based on the imaging capabilities of OCT and the biofilm imaging data obtained, OCT has potential to be used as a non-invasive, label-free, real-time, in-situ and/or in-vivo imaging modality for biofilm characterization.