Free-living amoebae (FLA) are protozoa that can be found in water networks where they prey on bacteria within biofilms. Most bacteria are digested rapidly by phagocytosis, however some are able to survive within amoebae and some are even able to multiply, as it is the case for Legionella pneumophila. These resisting bacteria are a potential health problem as they could also resist to macrophage phagocytosis. Several publications already reported intra-amoebal bacteria but the methods of identification did not allow metagenomic analysis and are partly based on co-culture with one selected amoebal strain. The aim of our study was to conduct a rRNA-targeted metagenomic analysis on amoebae and intra-amoebal bacteria found in drinking water network, to provide the first FLA microbiome in environmental strains. Three sites of a water network were sampled during four months. Culturable FLA were isolated and total DNA was prepared, allowing purification of both amoebal and bacterial DNA. Metagenomic studies were then conducted through 18S or 16S amplicons sequencing. Hartmannella was by far the most represented genus of FLA. Regarding intra-amoebal bacteria, 54 genera were identified, among which 21 were newly described intra-amoebal bacteria, underlying the power of our approach. There were high differences in bacterial diversity between the three sites. Several genera were highly represented and/or found at least in two sites, underlying that these bacteria could be able to multiply within FLA. Our method is therefore useful to identify FLA microbiome and could be applied to other networks to have a more comprehensive view of intra-amoebal diversity. Copyright © 2013 Elsevier Ltd. All rights reserved.