Availability of safe, pathogen-free drinking water is vital to public health; however, it is impossible to deliver sterile drinking water to consumers. Recent microbiome research is bringing new understanding to the true extent and diversity of microbes that inhabit water distribution systems. The purpose of this study was to determine how water chemistry in main distribution lines shape the microbiome in drinking water biofilms and to explore potential associations between opportunistic pathogens and indigenous drinking water microbes. Effects of disinfectant (chloramines, chlorine), water age (2.3 days, 5.7 days), and pipe material (cement, iron, PVC) were compared in parallel triplicate simulated water distribution systems. Pyrosequencing was employed to characterize bacteria and terminal restriction fragment polymorphism was used to profile both bacteria and eukaryotes inhabiting pipe biofilms. Disinfectant and water age were both observed to be strong factors in shaping bacterial and eukaryotic community structures. Pipe material only influenced the bacterial community structure (ANOSIM test, P < 0.05). Interactive effects of disinfectant, pipe material, and water age on both bacteria and eukaryotes were noted. Disinfectant concentration had the strongest effect on bacteria, while dissolved oxygen appeared to be a major driver for eukaryotes (BEST test). Several correlations of similarity metrics among populations of bacteria, eukaryotes, and opportunistic pathogens, as well as one significant association between mycobacterial and proteobacterial operational taxonomic units, provides insight into means by which manipulating the microbiome may lead to new avenues for limiting the growth of opportunistic pathogens (e.g., Legionella) or other nuisance organisms (e.g., nitrifiers).