Imaging mass spectrometry tools allow the two-dimensional visualization of the distribution of trace metals, metabolites, surface lipids, peptides and proteins directly from biological samples without the need for chemical tagging or antibodies, and are becoming increasingly useful for microbiology applications. These tools, comprising different imaging mass spectrometry techniques, are ushering in an exciting new era of discovery by enabling the generation of chemical hypotheses based on the spatial mapping of atoms and molecules that can correlate to or transcend observed phenotypes. In this Innovation article, we explore the wide range of imaging mass spectrometry techniques that is available to microbiologists and describe the unique applications of these tools to microbiology with respect to the types of samples to be investigated.