Two-photon (2P) microscopy has become increasingly popular among immunologists for analysing single-cell dynamics in tissues. Researchers are now taking 2P microscopy beyond the study of model antigen systems (e.g. ovalbumin immunization) and are applying the technique to examine infection in vivo. With the appropriate fluorescent probes, 2P imaging can provide high-resolution spatio-temporal information regarding cell behaviour, monitor cell functions and assess various outcomes of infection, such as host cell apoptosis or pathogen proliferation. Imaging of transgenic and knockout mice can be used to probe molecular mechanisms governing the host response to infection. From the microbe side, imaging genetically engineered mutant strains of a pathogen can test the roles of specific virulence factors in pathogenesis. Here, we discuss recent work that has applied 2P microscopy to study models of infection and highlight the tremendous potential that this approach has for investigating host-pathogen interactions.