Fifteen different cultivation media were used to isolate bacteria with the idea to obtain taxa specifically associated with ectomycorrhizae of <em>Tuber aestivum</em>. Ectomycorrhizae were collected at the sampling points previously analyzed for bacterial molecular diversity. We isolated 183 bacterial strains and identified them on the basis of the partial sequence of 16S rDNA. Out of these isolates, only 4 corresponded to operational taxonomic units significantly associated with <em>T. aestivum</em> ectomycorrhizae in previous molecular study. Preliminary study of the effect of 12 selected isolates on growth of T. aestivum mycelium showed no stimulation and one isolate induced the damage of hyphae. Different isolation strategy has to be developed to increase the probability of cultivation of potentially important components of <em>T. aestivum</em> mycorrhizosphere.