This paper describes a new microfluidic device called the “microfluidic palette”, capable of generating multiple spatial chemical gradients simultaneously inside a microfluidic chamber. The unique aspect of this work is that chemical gradients are generated by diffusion, without convection, and can either be held constant over long periods, or modified dynamically. We characterized a representative device with a 1.5 mm circular chamber where diffusion takes place, and three access-ports for the delivery and removal of solutes. The gradient stabilizes in ≈ 15 min for small molecules and can be maintained constant indefinitely. We demonstrate overlapping gradients with different spatial location and a controlled rotation of a diffusive gradient around its centre. Finally, we demonstrate the applicability of this tool to study the chemotactic response of the bacteria P. aeruginosa to glucose.