Clinical microbiology has changed considerably over the past decade with the advent of new mass spectrometric methods. The success of whole-cell MALDI-TOF mass spectrometry for rapid pathogen identification has markedly improved diagnostic analysis while effecting real cost savings. Currently, new approaches based on tandem mass spectrometry are emerging for identifying pathogens without the need for time-consuming culture steps. Such deep approaches are expected to, once again, revolutionize pathogen diagnostics. Nonetheless, the successes made in microbiological typing should not overshadow the opportunities that exist within the entire breadth of the potential application space! In my opinion, this special issue of Clinical Mass Spectrometry on “Microbiology and clinical diseases” is the perfect arena to begin exploration of these opportunities. Ideally, the reader will evaluate the strengths presented, realize the challenges and opportunities, and develop appreciation and strategies for future development; such is the progression of scientific advancement. In this special issue, we present publications that reflect the multi-faceted nature of applications of mass spectrometry in the clinical microbiology laboratory. Firmly established as the reference method for bacterial identification in routine practice, MALDI-TOF is now being extended towards identifying specific antibiotic resistance markers. Cordovana et al. [1] successfully explore such application on a set of common bacterial pathogens. Grenga et al. [2] review the most recent methodologies for pathogen proteotyping by mass spectrometry. Of note, tandem mass spectrometry-based proteotyping allows microorganism identification at an unprecedented specificity, even in complex samples, and, thus, should address the current method limits of whole-cell MALDI-TOF. Furthermore, such insightful proteotyping could provide additional phenotypic information, such as antibiotic resistance, on the pathogens present in the sample. Application of this proteomic approach for the analysis of human gut microbiota has been flourishing. Ngom et al. [3] review the potential of gut metaproteomics with a specific emphasis on combining other omics approaches, such as metagenomics and culturomics, to improve the reach of this area of research. There is little doubt that novel insights regarding human microbiota will be obtained. From this new knowledge, innovative diagnostic approaches will likely follow close behind. Multitudes of bacterial strains have acquired a high degree of resistance against antibiotics and only a few new antibiotic substances have been developed in recent years. It has become clear that the clinical prescription and outcome of antibiotic use must be optimized. Along this line, Zander et al. [4] propose an interesting viewpoint on the role of mass spectrometry in antibiotic stewardship. Of primary interest is improved monitoring to not only optimize treatment outcomes, but also reduce toxicity and the risk of emergence of acquired drug resistance. Kuhlin et al. [5] offer an interesting analysis of therapeutic drug monitoring in the context of tuberculosis, a worldwide infectious disease caused by Mycobacterium tuberculosis. In their review, the authors outline the pertinence of liquid chromatography coupled to tandem mass spectrometry for multiplex assays using low-volume human fluid samples. In this vein, Pautova et al. [6] deliver a new methodology for capturing microbial metabolites from blood serum prior to detection using mass spectrometry. Finally, Bregy et al. [7] show how metabolites from the headspace of saliva detected by mass spectrometry may help to diagnose and monitor periodontal diseases. I would like to thank all of the contributors for sharing their views and their latest research for this special issue. The community is also deeply in debt to the reviewers for taking the time to assess these contributions and provide helpful comments and numerous suggestions. Finally, I would like to thank the editorial board at Clinical Mass Spectrometry, especially Professor Michael Vogeser and Dr. Chris Herold, for giving me the opportunity to assemble this special issue. I hope that this compendium will encourage others to submit novel contributions to Clinical Mass Spectrometry and catalyze numerous discussions on the application of mass spectrometry in clinical microbiology.