Healthcare settings have been utilizing matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) since 2010. MALDI-TOF MS has various benefits over the conventional method of biochemical identification, including ease of use, speed, accuracy, and low cost. This approach can solve many of the obstacles to identifying bacteria, fungi and viruses. As technology advanced, more and more databases kept track of spectra, allowing species with similar morphological, genotypic, and biochemical traits to be identified. Using MALDI-TOF MS for identification has become more accurate and quicker due to advances in sample preparation and database enrichment. Rapid sample detection and colony identification using MALDI-TOF MS have produced promising results. A key application of MALDI-TOF MS is quickly identifying highly virulent and drug-resistant diseases. Here, we present a review of the scientific literature assessing the effectiveness of MALDI-TOF MS for locating clinically relevant pathogenic bacteria, fungi, and viruses. MALDI-TOF MS is a useful strategy for locating clinical pathogens, however, it also has some drawbacks. A small number of spectra in the database and inherent similarities among organisms can make it difficult to distinguish between different species, which can result in misidentifications. The majority of the time additional testing may correct these problems, which happen very seldom. In conclusion, infectious illness diagnosis and clinical care are being revolutionized by the use of MALDI-TOF MS in the clinical microbiology laboratory.