Host-pathogen interactions are highly regulated, dynamic processes that take place at the molecular, cellular and organ level. Innovative imaging technologies have emerged recently to investigate the underlying mechanisms of host-pathogen interactions. Innovations in fluorescence microscopy enable functional studies on the single-cell level. New light microscopes have been developed that improve the resolution to less than 100 nm. At the other extreme, intravital microscopy enables the correlation of cellular events on the organ level. This is also achieved by alternatives to microscopy such as bioluminescence, positron-emission tomography and magnetic resonance imaging. The methodologies described here will have a tremendous effect on our understanding of host-pathogen interactions.