Traditionally fungal identification has largely been based on subjective microscopic and macroscopic examination of morphological characteristics which requires extensive experience. In this study we have evaluated the potential of MALDI-TOF for rapid identification of filamentous fungi. 473 filamentous fungi representing 65 species from 42 genera were analysed either by extended formic acid extraction or the manufacturer’s recommended broth method. Spectra were acquired on the Bruker Microflex LT mass spectrometer and analysed against the Bruker filamentous fungi library v1.0. All isolates were run in duplicate and the identification was confirmed by either ITS sequencing and/or microscopic examination. Overall 387(82%) isolates were correctly identified by MALDI-TOF to the species level without considering log score values. 44 isolates were misidentified to the species level of which 42 isolates gave the correct genus. 42 isolates were unidentified. An unidentified isolate was defined as one which gave a poor acquisition by MS or, when examining the top 10 matches, there was no consistency in the species returned. Our laboratory has adopted a log score cut off of 1.7 and when this is applied 262 (55%) were correctly identified. However our experience has been that when the top 3 returned matches were the same species it is likely to be correct. This then provides useful information how to proceed with unidentified isolates by either looking for particular microscopic features, sub culturing to appropriate media or to proceed directly to sequencing. This approach has resulted in improved workflows, reduced turnaround times and assists meeting the challenges of an evolving workforce where skill levels vary and workload is rising. Our results indicate that integrating MALDI-TOF into a clinical mycology laboratory is advantageous. Adding organisms to an in house library, such as dematiaceous Hyphomycetes and Basidiomycetes, would greatly increase the numbers identified therefore further reducing the turnaround time associated with traditional methods.