The use of truffles in food is based mainly on the addition of artificial flavors, aiming to achieve an intense aroma in the products. As truffle is a natural product with nutritional and functional properties, it is important to find an optimal method for truffle storage. As the microbiota contribute to truffle aroma, the bacterial and yeast compositions in the rhizosphere and fruiting body of the truffle and the impact of different freezing methods on the volatile profile of the truffle Tuber aestivum during 90 days of the storage were determined. Bacteria and yeasts isolates were identified using 16s rRNA and 18s rRNA. The effect of freezing truffles at -20°C and -80°C with and without previous dipping in liquid N₂ on the volatile compounds was observed using GC/MS. The results demonstrated that the isolated bacteria belonged to the phylum Proteobacteria, Firmicutes and Actinobacteria, and the identified species mainly belonged to Firmicutes, genus Bacillus sp. Isolated yeasts were identified as Cryptococcus sp., Debaromyces hanseinii, Candida fermentati and Rhodotorula mucilaginosa. The GC/MS analysis revealed that frozen truffle samples were richer in the compounds 2-butanone, 2-methyl-butanal, methanethiol and 2-butanol after freezing or during storage. The content of DMS, acetaldehyde, 3-octanone, ethanol, and 2-methyl-1-propanol significantly decreased immediately after freezing. Overall, the gained results indicated that freezing of truffles as a preservation method had profound effects on the volatile compounds, while previous dipping in liquid N₂ showed no significant impact on the volatile profile of truffle Tuber aestivum.