Mass spectrometry (MS) methods furnish the clue to many microbiological applications including advanced studies on the diversity and classification of prokaryotes. Mass spectral data contribute to the polyphasic taxonomy which considers genotypic characters together with structure-functional and ecological traits. Additionally, these methods contribute to reliable and rapid identification of microorganisms bypassing conventional manipulations which are materials and time consuming. MS based analyses of biomarkers can be performed at the level of whole cells, cell homogenates, subcellular fractions, and individual molecules. For this purpose, various MS methods can be employed, such as MALDI-TOF, ESI, SELDI, and BAMS. Of these, MALDI-TOF MS is the especially easy-to-use and rapid method with many analytical applications, primarily in proteomics which aims at comprehensive description of protein inventory in prokaryotes. An alternative for detection and comparison of biomarkers via MS is amplification and alignment of marker gene sequences. Two molecular approaches supplement each other under support of database resources. Microbiologists readily assimilate MS methods propelled by high performance analyzers and sensitive detectors. The review focuses at progressing application of MS methods in microbiology, with an emphasis on identification and comparative study of bacteria.