Background <p>MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) technology has become an effective tool in clinical mycology laboratories in recent years for the identification of pathogenic filamentous fungi.</p> Methods <p>A total of 527 clinical specimens, comprising samples from both superficial and deep mycoses, were included in this study. Filamentous fungal samples isolated from various clinical specimens and identified using conventional methods between 2017 and 2018 at the Medical Microbiology Mycology Laboratory of Istanbul University-Cerrahpaşa Faculty of Medicine Hospital were analyzed using MALDI Biotyper Bruker Daltonik GmbH Revision 4 and the Filamentous Fungi database v3.0.</p> Results <p>In the identification of mold samples, the highest scoring identification values ​​were determined with the liquid cultivation and extraction method recommended by MALDI-TOF MS for filamentous fungi. A total of 46 different filament fungi species, including 6 dermatophytes and 40 non-dermatophyte filament fungi species, were identified from the clinical samples in the study. Of the total of 527 clinical samples, 27% (<i>n</i> = 142) were identified as having high reliability in the range of 1.8 to 3, and 20% (<i>n</i> = 103) as having low reliability in the range of 1.6 to 1.79. The total rate of identified filament fungi was successfully determined as 47% (<i>n</i> = 245).</p> Conclusions <p>In the practical experience of our medical mycology laboratory, we evaluated the current uses of MALDI-TOF MS and their practical application in daily routine. In the identification of mold species obtained from clinical samples by MALDI-TOF, it is important to meticulously prepare sample preparation protocols, use an identification strategy suitable for routine diagnosis, and continuously update libraries using advanced databases.</p>