Hot water plumbing is an important conduit of microbes into the indoor environment and can increase risk of opportunistic pathogens (for example, Legionella pneumophila). We examined the combined effects of water heater temperature (39, 42, 48, 51 and 58 degrees C), pipe orientation (upward/downward), and water use frequency (21, 3 and 1 flush per week) on the microbial composition at the tap using a pilot-scale pipe rig. 16S rRNA gene amplicon sequencing indicated that bulk water and corresponding biofilm typically had distinct taxonomic compositions (R-Adonis(2) = 0.246, P-Adonis = 0.001), yet similar predicted functions based on PICRUSt analysis (R-Adonis(2) = 0.087, P-Adonis = 0.001). Although a prior study had identified 51 degrees C under low water use frequency to enrich Legionella at the tap, here we reveal that 51 degrees C is also a threshold above which there are marked effects of the combined influences of temperature, pipe orientation, and use frequency on taxonomic and functional composition. A positive association was noted between relative abundances of Legionella and mitochondrial DNA of Vermamoeba, a genus of amoebae that can enhance virulence and facilitate replication of some pathogens. This study takes a step towards intentional control of the plumbing microbiome and highlights the importance of microbial ecology in governing pathogen proliferation.