The building plumbing microbiome has important implications, especially in terms of its role as a reservoir and conduit for the spread of opportunistic pathogens (OPs), such as Legionella pneumophila. This dissertation applied next-generation DNA sequencing tools to survey the composition of building plumbing microbiomes and assessed hypothetical factors shaping them. A challenge to identifying key factors shaping building plumbing microbiomes is untangling the relative contributions of influent water quality, provided by drinking water utilities, and those of building-level features, such as pipe materials. To this end, standardized pipe rigs were deployed at the treatment plants and in distal portions of the water distribution system at five water utilities across the eastern U.S. Source water and treatment practices appeared to be the overarching factors shaping the microbial taxonomic composition at the tap, with five key water chemistry parameters identified (total chlorine, pH, P, SO42- and Mg2+). Hot water plumbing is of particular interest because OPs tend to proliferate in warm water environments and can be inhaled in aerosols when showering. Two identical lab-scale recirculating hot water rigs were operated in parallel to examine the combined effects of water heater temperature set point, pipe orientation, and water use frequency on the hot water plumbing microbiome. Our results revealed distinct microbial taxonomic compositions between the biofilm and water phases. Importantly, above a threshold of 51 °C, water heater temperature, pipe orientation, and water use frequency together incurred a prominent shift in microbiome composition and L. pneumophila occurrence. While heat shock is a popular means of remediating L. pneumophila contamination in plumbing, its broader effects on the microbiome are unknown. Here, heat shock was applied to acclimated lab-scale hot water rigs. Comparison of pre- versus post- heat shock samples indicated little to no change in either the microbial composition or L. pneumophila levels at the tap, where both water heater temperature and water use frequency had the most dominant effect. Overall, this dissertation contributes to advancing guidance regarding where to most effectively target controls for OPs and also advances research towards identifying the features of a 'healthy' built environment microbiome. / PHD / Drinking water is often misconceived to be “sterile,” whereas in reality the water distribution and plumbing systems that convey the water to the consumer represent a robust microbial habitat. While it is not possible, or even desirable, to kill all of the microbes present in drinking water, the Safe Drinking Water Act in the U.S. enforces measures to purify and disinfect water at the treatment plant and keep bacterial numbers low in water mains and up to the consumer property line. However, current regulatory frameworks are designed to protect against fecal- (e.g., raw sewage and manure) derived pathogens, whereas recently opportunistic pathogens (OPs), including Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa have come to the forefront as the leading source of tap-water related illness in the U.S. and other developed countries. In contrast to traditional fecal pathogens, building plumbing systems are a natural habitat for OPs, where they can readily proliferate. Currently there are no provisions within the Safe Drinking Water Act or other regulations to protect consumers specifically from OPs. There are also no “silver bullet” remedial measures that consistently and reliably defend against OPs colonizing building building plumbing, particularly when aiming to protect against multiple types of OPs. A major challenge in preventing and remediating OP proliferation in building plumbing is that they tend to be protected from disinfectants, such as chlorine, inside amoeba hosts and within the slimy layer that forms on the surface of pipe walls called “biofilm”. With the recent advent over the past decade of next-generation DNA sequencing, there are new reasons to take interest in the microbial composition of tap water. In particular, next-generation DNA sequencing has provided new insight into the composition of the human microbiome, e.g., the microbes that naturally inhabit our skin, gut, and lungs, and has revealed striking relationships with human health (e.g., obesity, diabetes, asthma, autism, allergies). The question naturally arises with respect to the factors shaping the human microbiome, with role of the “built environment” being of fundamental interest. The built environment; including homes, offices, schools, hospitals, and vehicles, is where most humans in developed countries spend > 90% of their time. Tap water is likely an important feature shaping the microbiome of the built environment, serving as a conduit for microbes into tiny droplets called aerosols, which can be inhaled into the lungs or otherwise inoculate the skin during showering or be transferred onto food during food preparation. Thus, there is interest in mapping out the microbiome of tap water and the factors that shape it, not only because of its potential to harbor OPs, but because of its potential general effect on built environment and human microbiomes. Long-term research could lead towards identifying which microbes serve a beneficial, or “probiotic,” role in preventing pathogen growth and benefiting human health. The purpose behind the body of research described in this dissertation was to apply newly available next-generation DNA sequencing tools towards mapping out the microbial composition characteristic of tap water, with emphasis on implications for preventing proliferation of OPs. Of particular interest was the relative role of what water utilities and building operators can do to protect public health. To this end, the DNA sequencing approach was applied to carefully controlled and replicated field- and laboratory-scale plumbing rigs to gain insight into the relative roles and interactions of the water quality provided by drinking water utilities and practical building-level engineering controls. Specific factors investigated included: stagnation (i.e., the tendency of water to sit unused in pipes in 8 hour cycles), pipe material (e.g., metallic versus plastic), pipe configuration (i.e., up or down flow to induce convective mixing vs stratification, respectively), water heater temperature set point (i.e., balancing hotter temperatures needed to kill pathogens versus lower temperatures desirable to save energy or prevent scalding), and heat-shock treatment (i.e., temporarily elevating the water heater temperature and flushing the system to kill off pathogens). There were several general findings that can be highlighted based on this research. First, based on comparison of standardized plumbing rigs installed at five water utilities in the U.S., the nature of the water provided by the local water utility was the overarching factor shaping the microbiome composition at the tap, moreso than pipe material or stagnation. Second, there exists an ideal threshold water heater temperature setting (51 °C based on the conditions of this study) above which there is a concordant shift in microbiome composition and decrease in L. pneumophila occurrence. Third, consistent water heater temperature setting above this threshold has a stronger long-term influence on the microbiome composition and L. pneumophila control than temporarily elevating the temperature for heat-shock treatment. Finally, biofilm and bulk water microbial compositions are extremely diverse in composition (e.g., thousands of species of microbes in each) and functional markers, and distinct from one anaother in terms of their characteristics under different operational conditions. In sum, this study takes a step towards better understanding building plumbing microbiome and identifies several promising engineering and control factors that can ultimately inform intentional engineering of the building plumbing microbiome, particularly with respect to protecting public health against OPs and potentially other microbiome-related ailments in the future.