Lab-on-a-chip platforms present many new opportunities to study bacterial cells and cellular assemblies. Here, a new platform is described that allows application of uniaxial stress to individual bacterial cells while observing the cell and its subcellular assemblies using a high resolution optical microscope. The microfluidic chip consists of arrays of miniature pressure actuated valves. By placing a bacterium under one of such valves and partially closing the valve by externally applied pressure, the cell can be deformed. Although large pressure actuated valves used in integrated fluidic circuits have been extensively studied previously, here those microfluidic valves are downsized and flow channels with rectangular cross-sections are used to maintain the bacteria in contact with cell culture medium during the experiments. The closure of these valves has not been characterized before. First, these valves are modeled using finite element analysis, and then the modeling results are compared to the actual closing profiles of the valves, which is determined from absorption measurements. The measurements and modeling show with good agreement that the deflection of valves is a linear function of externally applied pressure and the deflection scales proportionally to the width of the flow channel. In addition to characterizing the valve, the report also demonstrates at a proof-of-principle level that the device can be used to deform a bacterial cell at considerable magnitude. The largest deformations are found in 5 μm wide channels where the bacterial width and length increase by 1.6 and 1.25 times, respectively. Narrower and broader channels are less optimal for these studies. The platform presents a promising approach to probe, in a quantitative and systematic way, the mechanical properties of not only bacterial cells but possibly also yeast and other single-celled organisms.