A large number of bacterial species move smoothly on solid surfaces in the absence of extracellular -organelles. In the deltaproteobacterium Myxococcus xanthus, this surface motion, termed gliding motility, involves a novel macromolecular machinery Agl-Glt. During the motility process, the Agl-Glt system, an integral envelope protein complex, is assembled on the ventral side of the cell. Doing so, the complex couples surface adhesion to the activity of the Agl motility motor. On the cytosolic side, the Agl-Glt system is linked to the bacterial actin cytoskeleton MreB. It is proposed that motility is produced when surface immobilized Agl-Glt complexes produce traction on a rigid track, possibly the MreB cables. Testing this hypothesis directly requires both microfluidic techniques to perturb the motility process with inhibitors (i.e., A22, CCCP) and state-of-the-art microscopy techniques (i.e., TIRF and AFM). These approaches require a microscopy chamber where the cells glide in liquid on a non-agar substrate. Here, we describe a straightforward coating procedure to construct a chitosan-functionalized microfluidic chamber that fulfills these requirements. This set up circumvents all the disadvantages of traditional agar-based assays, providing new grounds for high-resolution experiments. We also describe simple image processing to maximize the quality of data representation. In theory, our procedure could be used for any bacterial system that adheres to chitosan.