Background and aims Plant seeds are emerging micro–habitats, whose importance as reservoir and vector of beneficial microbes just begins to be recognized. Here we aimed to characterize the bacterial microbiota of the Anadenanthera colubrina seed endosphere, with special focus to beneficial traits and to the colonization pattern. Methods Cultivation–dependent (isolation from surface–sterilized seeds) and cultivation–independent (pyrosequencing of 16S rRNA gene from metagenomic seed DNA) analyses, functional tests and microscopical investigations (fluorescence in situ hybridization coupled with confocal laser scanning microscopy (FISH-CLSM) were performed. Results We isolated several Methylobacterium and Staphylococcus spp., exhibiting both plant growth promotion and antimicrobial activities. The two taxonomic groups showed complementary traits, which supports a functional selection. Both genera were detected also by pyrosequencing, together with further taxa. The genera Friedmaniella, Bifidobacterium, Delftia, Anaerococcus and Actinomyces appeared here for the first time as seed endophytes. We detected bacterial cells and micro–colonies in seed cryosections by FISHCLSM. Alphaproteobacteria, Firmicutes and other bacteria colonized intercellular spaces of the parenchyma and associated to transport vessels. Conclusions This work sheds light onto the diversity, functions and colonization pattern of the Anadenanthera colubrina seed endophytes, and strongly suggest a role as beneficial partners for seed-associated microbiota