Bacteria are ubiquitous in irrigation water resources and can include pathogens that may compromise food safety and public health. However, only a small fraction of total bacterial community members in water can be identified through standard culture-based laboratory methods. 16S rRNA and shotgun sequencing techniques have improved the identification of non-culturable bacteria in water resources. Nevertheless, because sequencing approaches are nucleic-acid based, they are unable to differentiate between the proportion of bacterial communities identified that are live and metabolically-active versus those that are represented by free, relic DNA, not present in viable cells. To bridge this knowledge gap, my dissertation research coupled DNA-labeling (using 5-bromo-2’- deoxyuridine (BrdU) and propidium monazide (PMA)) with next-generation sequencing approaches to identify and comprehensively characterize metabolically-active bacteria in multiple nontraditional irrigation water sources in the Mid-Atlantic region. My aims were as follows: 1) To characterize the metabolically-active fraction of bacterial communities, as well as antibiotic resistance genes and virulence gene profiles in nontraditional irrigation water sources; 2) To evaluate culture-dependent and -independent methods in the detection of metabolically-active pathogenic and non-pathogenic Vibrio species in four nontraditional irrigational water sources; and 3) To track metabolically-active bacterial communities from rooftop-harvested rainwater to irrigated produce in Maryland. Overall, we identified diverse metabolically-active bacterial communities in all nontraditional water sources. Notably, we observed the presence of viable bacteria of importance to both human and/or animal health (Actinobacterium spp., Flavobacterium spp., Aeromonas spp. Pseudomonas spp. and Vibrio spp.). Interestingly, diverse antimicrobial resistance and virulence genes were predominantly found in non-BrdU-treated samples, indicating that these genes can persist in relic DNA and could be transferred to other environmental bacteria through transformation events. We also source-tracked viable bacteria, including Sphingomonas spp., Enterobacter spp., Enterococcus spp, and Citrobacter spp. from rooftop-harvested irrigation water to produce. In summary, this work provides the first description of total, viable, and metabolically-active bacterial communities in different nontraditional irrigation water sources. These data can be used to improve risk characterization of these water sources, and ultimately inform the selection of appropriate cost-effective remediation methods to treat these waters prior to irrigation activities in order to prevent foodborne outbreaks.