Aims Compare bacterial communities in non-vegetated soils and in the rhizosphere of Ni-hyperaccumulating or Ni-excluding plants from four serpentine sites of the Iberian Peninsula. Methods Bacterial communities in non-vegetated soil and in rhizosphere of Alyssum serpyllifolium, and Dactylis glomerata, were analysed using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments of the total bacterial community and of Alphaproteobacteria, Actinobacteria and Streptomycetaceae. Results Rhizosphere bacterial communities of, either the hyperaccumulators or the excluder plants, were significantly different from communities in non-vegetated soils, especially for Alphaproteobacteria and Actinobacteria. The main differences between the rhizobacterial communities of the hyperaccumulator and of the excluder corresponded to Alphaproteobacteria profiles. Communities in non-vegetated soil were significantly influenced by intrinsic soil properties while in rhizosphere soil these factors were replaced by parameters related to nutrient availability. Members of the genera Blastococcus, Geodermatophilus, Modestobacter, Rhodococcus, Pseudonocardia, Devosia and Hyphomicrobium were detected in the rhizosphere of the Ni-hyperaccumulating plants. Conclusions Bacterial communities significantly differed between hyperaccumulator and excluder rhizosphere and non-vegetated soil. Actinobacteria of the Geodermatophilaceae family and Alphaproteobacteria of the Hyphomicrobiaceae family predominated in the rhizosphere profiles of the Ni-hyperaccumulators. Alphaproteobacteria could participate in the tolerance of Poaceae to trace elements in ultramafic soils.