Truffles (Tuber melanosporum) are high value commercial fungi traditionally produced in the northern hemisphere. There is little understanding of the ecology of truffle bacterial microbiome and the role it plays in aroma development and spoilage during post-harvest storage. The aim of this study was to investigate the population structure and the functional potential of the bacterial communities associated with black perigord truffles from Spain using metagenomics and gas chromatography mass spectrometry. Black perigord truffle samples (N=14) were provided by Australian Truffle Traders, Manjimup, Western Australia during the Spanish truffle season. The fresh truffle samples were assessed at 3 time intervals, 0, 7 and 14 days to monitor the change in the bacterial population during storage. The V3-V4 region of 16s rRNA DNA was sequenced using the Illumina MiSeq Platform using paired end reads. Alpha-diversity of the Spanish truffles was low, with Gammaproteobacteria and Alphaproteobacteria dominating the populations. Operational taxonomic units relating to Enterobacteriaceae, Rhizobiaceae, and Bradyrhizobiaceae families were the most abundant sequences in the samples. Previous studies have also found Rhizobiales dominate across truffle species and geographic regions. The results in this study add further evidence to the possibility that truffles may form a mutualistic relationship with Rhizobiales, with the bacteria able to fix atmospheric nitrogen in the truffle fruiting bodies. Additionally, dimethyl sulphide compounds produced by Enterobacteriaceae may explain the high concentrations of DMS found in our GC-MS analysis of the truffles. Similar analysis is currently being performed with Australian black perigord truffles. It is hoped that by comparing data from the two regions this will allow truffle growers an insight into how the microbiome of the region may influence the growth and aromatic qualities of their truffles and the impact these microbes have on truffle spoilage.