Fried ripened curd cheese is a traditional dairy product from the Wielkopolska region of Poland. It is made from cows' milk and has Protected Geographical Indication (PGI) status. The aim of this study was to use the microbial-culture-independent PCR-DGGE technique to examine whether a link exists between the fungal community present in the product and the geographical origin of the cheese. To look for correlations between the geographical origin and the microbiota composition, the DGGE fingerprinting profiles were subjected to a multivariate analysis, which included k-means clustering and principal component analysis (PCA). The dominant eukaryotic community of this traditional product, which has been investigated here for the first time, was shown to be composed of Galactomyces geotrichum, Kluyveromyces marxianus, Pichia kudriavzevii, Candida inconspicua, and Trichosporon spp. Our results demonstrated that PCR-DGGE is a simple and relatively inexpensive method for the traceability of Wielkopolska fried cheese and could be extended to other microbe-enriched foodstuffs.