Several non-culture molecular (multiplex polymerase chain reaction assays, DNA microarrays, massive parallel DNA sequencing, in situ hybridization, microbiome profiling, and molecular typing of pathogens) and analytical (electrophoresis, gel electrophoresis, surface-enhanced Raman scattering, and mass spectrometry) tools have been developed in recent years for the identification of bacteria and diagnosis of bacterial infections from clinical samples. Among mass spectrometric techniques, electrospray ionization (ESI) and rapid evaporative ionization (REI) mass spectrometric (MS) techniques have attracted much attention in the identification of microorganisms (bacteria, fungi, and viruses), and in the diagnosis of various bacterial infections. This review highlights the developed ESI-MS-based methods, including polymerase chain reaction (PCR) combined with ESI-MS and capillary electrophoresis (CE) and liquid chromatography (LC)-ESI-MS, for the identification of microorganisms (pathogenic bacteria, fungi, and viruses) in various samples. Recent applications of ESI- and REI-MS in identifying pathogenic bacteria are depicted in tables, and some significant findings are summarized.