Single-cell imaging of host-microbe interactions over time is impeded by cellular motility because the cells under scrutiny tend to migrate out of the imaging field. To overcome this technical challenge, we developed a microfluidic platform for imaging hundreds of individual motile phagocytic cells and bacteria within microfluidic traps that restrict their movement. The interaction of trapped host cells and bacteria is monitored by long-term time-lapse microscopy, allowing direct visualization of all stages of infection at the single-cell level. The medium flowing through the microfluidic device can be changed quickly and precisely, permitting the real-time imaging of cellular responses to antibiotics or other environmental stresses. Here, we demonstrate the potential applications of this approach by co-culturing the phagocytic amoeba Dictyostelium discoideum with the intracellular pathogen Mycobacterium marinum. However, the platform can be adapted easily for use with other host cells or microorganisms. This approach will provide new insights into host-pathogen interactions that cannot be studied using conventional population-based assays.