Next generation sequencing offers several ways to study microbial communities. For agri-food sciences, identifying species in diverse food ecosystems is key for both food sustainability and food security. The aim of this study was to compare metabarcoding pipelines and markers to determine fungal diversity in food ecosystems, from Illumina short reads. We built mock communities combining the most representative fungal species in fermented meat, cheese, wine and bread. Four barcodes (ITS1, ITS2, D1/D2 and RPB2) were tested for each mock and on real fermented products. We created a database, including all mock species sequences for each barcode to compensate for the lack of curated data in available databases. Four bioinformatics tools (DADA2, QIIME, FROGS and a combination of DADA2 and FROGS) were compared. Our results clearly showed that the combined DADA2 and FROGS tool gave the most accurate results. Most mock community species were not identified by the RPB2 barcode due to unsuccessful barcode amplification. When comparing the three rDNA markers, ITS markers performed better than D1/D2, as they are better represented in public databases and have better specificity to distinguish species. Between ITS1 and ITS2, differences in the best marker were observed according to the studied ecosystem. While ITS2 is best suited to characterize cheese, wine and fermented meat communities, ITS1 performs better for sourdough bread communities. Our results also emphasized the need for a dedicated database and enriched fungal-specific public databases with novel barcode sequences for 118 major species in food ecosystems.