Background: Pathogenic moulds are an important cause of morbidity and mortality especially among immunocompromised patients with diabetes, after organ transplant, AIDS, and leukemia. Therefore, rapid, and accurate identification of moulds is strongly needed. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI TOF MS) has become a powerful, fast, and widely used tool for microorganisms' identification in most sophisticated laboratories around the world. MALDI TOF MS utilizes the analysis of the ribosomal proteins by generating unique spectrum for each microorganism. This study aims at expanding the use of MALDI-TOF MS by establishing its use for mould identification in the Microbiology and Immunology laboratory, Sultan Qaboos University Hospital (SQUH). Methods: A total of 71 isolates representing (12) genera and (26) species were used in this study. Extraction (Ext) and liquid cultivation (LC) for identification by MALDI-TOF MS were used for sample preparation. In addition, the identification by MALDI-TOF MS was compared with the molecular identification for all isolates. Results: Based on Bruker's criteria, the species identification rate was 43.7% and 26.8% by LC and Ext methods, respectively. The genus identification rate was 97.2% and 93% by LC and Ext methods, respectively. The cut-off value was lowered for species identification to ≥1.7 (adjusted criteria) which significantly increased the species identification rate to 78.9% by both methods, whereas misidentification rate increased at species level only. The genus identification rate was 100% for Aspergillus and dermatophytes isolates by both methods and based on both criteria. Based on adjusted criteria, Aspergillus species identification rate increased significantly by both methods to 93 % without occurrence of misidentification at species level. On the other hand, dermatophytes species identification rate increased to 50% by both methods based on adjusted criteria with high increase in misidentification rate (at species level only). Misidentification observed with Trichophyton species was either due to lack of reference spectra in the used database or due to the known limitation of Bruker MALDI-TOF MS to differentiate between Trichophyton species that have similar patterns of spectra. For Mucorales, LC method significantly increased the genus identification rate to 81.8% based on Bruker criteria. Based on adjusted criteria, Mucorales species identification rate increased by both methods to 45.5% with increase in misidentification rate at species level only. The reproducibility of MALDI-TOF MS by both methods was 100%. The genus identification accuracy for MALDI-TOF MS was 100% by both methods based on both criteria. However, at the species level, identification accuracy by both methods based on both criteria was less than the acceptable percentage. Conclusion: Bruker MALDI-TOF MS in Microbiology diagnostic laboratory in SQUH can be used for moulds identification in conjunction with phenotypic identification method and limitations should be considered. Ext method with adjusted criteria can be used for Aspergillus species and dermatophytes genus identification. In contrast to Aspergillus and dermatophytes, LC method and adjusted criteria can be used for Mucorales genus identification.