Abstract: The incidence of invasive fungal infection (IFI) increased significantly in the past few years due to the increase of immunocompromised patients. The morbidity and mortality of IFI are high, and the diagnosis and treatment of IFI are usually difficult. It is estimated that the mortality rate of IFI exceeds 50.00% and approximately 1.5 million deaths can be caused by IFI annually. The identification of filamentous fungi in the clinical laboratory is usually based on morphological and molecular biological examination of cultured colonies isolated, which is a slow and complex process. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a simple, rapid, efficient, high-throughput, and low-cost method and can be used in the routine identification of microorganisms in clinical microbiology laboratories. Nowadays, MALDI-TOF MS has become the mainstream technique for bacterial identification in most clinical laboratories. However, its application in the identification of filamentous fungi is not wide, which is mainly attributed to the lack of standardized protocol for protein extraction and limited fungus databases. In addition, there is limited research on the factors affecting the accuracy of MALDI-TOF MS identification of pathogenic fungi. This paper describes and analyzes the factors affecting the accuracy of MALDI-TOF MS identification of pathogenic fungi, focusing on the impacts of different culture conditions and databases.