During the last two decades our understanding of the complex in vivo host-pathogen interactions has increased due to technical improvements and new research tools. The rapid advancement of molecular biology, flow cytometry and microscopy techniques, combined with mathematical modelling, have empowered in-depth studies of systemic bacterial infections across scales from single molecules, to cells, to organs and systems to reach the whole organism level. By tracking subpopulations of bacteria in vivo using molecular or fluorescent tags, it has been possible to reconstruct the spread of infection within and between organs, allowing unprecedented quantification of the effects of antimicrobial treatment and vaccination. This review illustrates recent advances in the study of heterogeneous traits of the infection process and illustrate approaches to investigate the reciprocal interactions between antimicrobial treatments, bacterial growth/death as well as inter- and intra-organ spread. We also discuss how vaccines impact the in vivo behaviour of bacteria and how these findings can guide vaccine design and rational antimicrobial treatment.