It has been suspected that some species belonging to the alphaproteobacteria might cause pneumonia in humans. It is thus of special interest to isolate new members of this phylum, and to further characterize their pathogenicity. The amoebal co-culture method allowed the isolation of various new bacterial species during the last few years, including fastidious alphaproteobacterial species that were isolated from complex environments. In this work, we isolated new bacterial strains from a drinking water network or from river water using amoebal co-culture with Acanthamoeba castellanii. One Afipia sp. strain and two Bosea sp. strains presented 16SrDNA and partial rpoB gene sequences suggesting that they could be representative of new species, and were thus further characterized using phenotypic tests.