AbstractStudying the events that occur when a pathogen comes into contact with its host is the basis of the field of infection biology. Over the years, work in this area has revealed many facets of the infection process, including attachment, invasion and colonization by the pathogen, and of the host responses, such as the triggering of the immune system. Recent advancements in imaging technologies, such as multiphoton microscopy (MPM), mean that the field is in the process of taking another big leap forward. MPM allows for cellular‐level visualization of the real‐time dynamics of infection within the living host. The use of live animal models means that all the interplaying factors of an infection, such as the influences of the immune, lymphatic and vascular systems, can be accounted for. This review outlines the developing field of MPM in pathogen‐host interactions, highlighting a number of new insights that have been ‘brought to light’ using this technique.