One of the main objectives of the BCCM/IHEM fungal culture collection is the long-term preservation of interesting strains to ensure their availability for the scientific community at large. The viability, purity and identity of a strain is verified before entering the collection, but also after lyophilisation or cryopreservation. For filamentous fungi, these routine identity checks are traditionally done based on morphological analyses, and DNA sequencing if necessary. This requires extensive taxonomical expertise, and is time consuming and costly. The BCCM/IHEM collection evaluated matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) as an alternative method for the routine post-preservation identity controls. A total of 481 controls were carried out using MALDI-TOF MS in parallel with the conventional procedure. The overall performance of the MALDI-TOF MS reached 84% of correct identifications at species level. Moreover, misidentification at entry in the collection could be corrected for 14 strains by mass spectrometry, which was confirmed by DNA sequencing. Out of these 14, only eight had been detected by the traditional method. Considering these results, a workflow combining MALDI-TOF MS, microscopy and genetic analyses is proposed to enhance accuracy and time- and cost-effectiveness of routine identity controls in fungal culture collections.