Single-cell analysis uncovers phenotypic differences between cells in a population and dissects their individual physiological states and differences on all omics levels from genome to phenome. Spectrometric observation allows label-free analysis of the metabolome and proteome of individual cells, but is still mainly limited to the analysis of mammalian single cells. Recent progress in mass spectrometry approaches now enables the analysis of microbial single cells — mainly by miniaturizing cell handling, incubation, and improving chip-coupling concepts for analyte ionization by interfacing microfluidic chips and mass spectrometers. This review aims at distilling the enabling principles behind microbial single-cell mass spectrometry and puts them into perspective for the future of the field.