Fungal diseases have an increasing global impact on human and animal health. Invasive fungal diseases alone cause over 1.6 million deaths/year. To reduce this burden advancements in diagnoses are essential. DNA barcoding has recently been established as the gold-standard identification technique for pathogenic fungi. This method relies on the selection of short, standardized DNA regions - DNA barcodes - that are divergent at the species level. For pathogenic fungi, the primary barcode (internal transcribed spacer region) and secondary barcode (translational elongation factor 1α) have been established, and together form the dual barcoding scheme. Clinical implementation of these barcodes relies on databases of quality-controlled reference sequences, e.g. the “ISHAM Barcoding Database”, aiming to provide reference sequences for both fungal barcodes. The current study aimed to generate secondary fungal barcode reference sequences to complement the primary fungal barcode, and to evaluate the discriminatory power of the dual barcoding scheme. As a result, 270 reference sequences were generated from 90 pathogenic fungal species. To evaluate the dual barcoding scheme, barcoding gap analysis of each barcode and in combination was performed for select fungal taxa. Barcoding gap analysis revealed barcoding gaps when using the secondary barcode for all taxa where the primary barcode was unable to do so. For all taxa analysed the combination of the two barcodes revealed barcoding gaps. These results indicate that either the secondary barcode alone or in combination with the primary barcode enables accurate identification of the studied fungal species. As such, the dual barcoding scheme in combination with the extension of the “ISHAM Barcoding Database” establishes a highly accurate and rapid identification system for fungal pathogens for routine diagnostics of mycoses.