High quality freshwater is a vital resource for sustaining agriculture and feeding a growing global population. Yet, due to increasing declines in groundwater, key food production regions across the world face uncertainty with regard to water availability. Nontraditional irrigation water sources, such as reclaimed water (advanced treated municipal wastewater) and untreated surface water (e.g. creeks, ponds, and brackish rivers), may contribute to sustainable solutions to conserve groundwater supplies. However, the microbial community composition and dynamics within these water sources are typically poorly characterized and comparative analysis of their microbial communities are rare. Using high-throughput, cultivation-independent sequencing methodologies, this dissertation research focused on three aims: 1) exploring the functional and taxonomic features of bacteria in nontraditional irrigation water sources; 2) assessing the bacterial and viral communities of agricultural pond water in relation to seasonality; and 3) describing the dynamics, composition, and potential dissemination of irrigation water microbiota from a freshwater creek to an irrigated field. The first aim was addressed through a broad investigation of bacteria within agricultural ponds, freshwater creeks, brackish rivers, and reclamation facilities. Through metagenomic-based analyses, features of the bacterial community, such as antimicrobial resistance genes (ARGs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) arrays, were found to vary by sampling date and specific site. For the second aim, agricultural pond water was sampled over two time periods and found to harbor diverse bacteria and bacteriophage species, the abundance and composition of which were influenced by factors characteristic of the pond’s topography and seasonality. For the final aim, samples from a creek used actively for agricultural irrigation, as well as samples of pre- and post-irrigated soil, were analyzed. ARGs and virulence factors were identified in the water and soil samples, with the majority being specific to their respective environment. Moreover, analyses of CRISPR arrays from the creek samples indicated the persistence of certain bacterial lineages, as well as specific interactions between creek bacteriophage and their hosts. Overall, this research improves scientific knowledge of bacterial and viral composition, dynamics, and interactions that can be utilized to assess the suitability and safety of nontraditional irrigation water sources.