In order to understand the microbial communities in drinking water biofilms, both eukaryotic and bacterial communities in three faucet biofilms were characterized by 454 pyrosequencing and quantitative PCR approaches. Microbial assemblages of the biofilms were dominated by bacteria, with Sphingomonadales, Rhizobiales, and Burkholderiales comprising the major bacterial populations. Although about 2 years of biofilm development occurred, the microbial community at site WSW still demonstrates the characteristics of a young biofilm community, e.g. low biomass, abundant aggregating bacteria (Blastomonas spp. and Acidovorax spp.) etc. Hartmannella of amoebae was the dominant eukaryotic predator in the biofilms, and correlated closely with biofilm bacterial biomass. Nonetheless, there was no obvious association of pathogens with amoebae in the faucet biofilms. In contrast, residual chlorine seems to be a dominant factor impacting the abundance of Legionella and Mycobacterium, two primary potential opportunistic pathogens detected in all faucet biofilms.