We report the isolation and characterization of endophytic bacteria, endemic to serpentine outcrops of Central Italy, from a nickel hyperaccumulator plant, Alyssum bertolonii Desv. (Brassicaceae). Eighty-three endophytic bacteria were isolated from roots, stems, and leaves of A. bertolonii and classified by restriction analysis of 16S rDNA (ARDRA) and partial 16S rDNA sequencing in 23 different taxonomic groups. All isolates were then screened for siderophore production and for resistance to heavy metals. One isolate representative of each ARDRA group was then tested for plant tissue colonization ability in sterile culture. Obtained results pointed out that, despite the high concentration of heavy metals present in its tissues, A. bertolonii harbors an endophytic bacterial flora showing a high genetic diversity as well as a high level of resistance to heavy metals that could potentially help plant growth and Ni hyperaccumulation.