Cell motility and chemotaxis play a key role in many essential biological processes including wound healing, cancer metastasis, and embryonic development. We present recent work that addresses motility in both bacterial and eukaryotic systems. On the one hand, we analyze the trajectories of moving cells to characterize swimming behavior and wall interactions and to derive statistical descriptions for cell motion in terms of Langevin‐type equations. On the other hand, we study the intracellular dynamics of the actin cytoskeleton in motile cells that are exposed to different patterns of chemical stimuli. A combined approach of fluorescence imaging, microfluidics, and photo‐uncaging provides a high level of spatiotemporal control to study the response of single chemotactic cells of the social amoeba Dictyostelium discoideum.