Microbes influence many aspects of human life from the environment to health, yet evaluating their biological pro- cesses at the chemical level can be problematic. Mass spectrometry imaging (MSI) enables direct evaluation of microbial chem-ical processes at the atomic to molecular levels without destruction of valuable two-dimensional information. MSI is a label-free method that allows multiplex spatiotemporal visualization of atomic- or molecular-level information of microbial and microbe-related samples. As a result, microbial MSI has become an important field for both mass spectrometrists and microbiologists. In this review, basic techniques for microbial MSI, such as ionization methods and analyzers, are explored. In addition, we discuss practical applications of microbial MSI and various data-processing techniques.