Low permeability regions such as clay lenses are difficult to remediate using conventional treatment methods. Bacterial chemotaxis (directed migration toward a contaminant source) may be helpful in enhancing bioremediation of such contaminated sites. This study experimentally simulates a two-dimensional dual-permeability groundwater contamination scenario using a microfluidic device (MFD) and evaluates transverse chemotactic migration of bacteria from high to low permeability regions under various flow velocities. Chemotaxis of Escherichia coli (E. coli) HCB33 to the chemoattractant dl-aspartic acid was quantified in terms of change in total bacterial counts in pore throats in low permeability regions containing attractant. An increase in total bacterial counts, ranging from 1.09 to 1.74 times, was observed in low permeability regions in experiments under chemotactic conditions. Experiments with no attractant showed no increase in total bacterial counts in low permeability regions. A large increase in bacterial counts in the pore throats just outside the low permeability region was also observed in chemotaxis experiments. The bacterial chemotactic response was observed to decrease linearly with increase in flow velocity, with no observed response at the highest flow velocity (Darcy velocity = 0.22 mm/s), where chemotaxis was offset by advective flow.