Manure from livestock production has been associated with the contamination of water resources. To date, research has primarily focused on runoff of these contaminants from animal operations into surface water, and the introduction of poultry-derived pathogenic zoonoses and other contaminants into groundwater is under-investigated. We characterized pathogens and other microbial and chemical contaminants in poultry litter, groundwater, and surface water near confined poultry feeding operations (chicken layer, turkey) at 9 locations in Iowa and one in Wisconsin from May and June 2016. Results indicate that poultry litter from large-scale poultry confined feeding operations is a likely source of environmental contamination and that groundwater is also susceptible to such poultry-derived contamination. Poultry litter, groundwater, and surface water samples had detections of viable bacteria growth (Salmonella spp., enterococci, staphylococci, lactobacilli), multi-drug resistant Salmonella DT104 flost and int genes, F+ RNA coliphage (group I and IV), antibiotic resistance genes (ARGs; blaDHA, blaOXA-48, blaTEM, blaCMY-2, tetM), phytoestrogens (biochanin A, daidzein, formononetin), and a progestin (progesterone). In addition, mcr-1 (a colistin ARG), was detected in a groundwater sample and in another groundwater sample, antibiotic resistant isolates were positive for Brevibacterium spp., a potential signature of poultry in the environment. Detectable estrogenicity was not measured in poultry litter, but was observed in 67% of the surface water samples and 22% were above the U.S. Environmental Protection Agency trigger level of 1 ng/L. The transport of microbial pathogens to groundwater was significantly greater (p < 0.001) than the transport of trace organic contaminants to groundwater in this study. In addition to viable pathogens, several clinically important ARGs were detected in litter, groundwater, and surface water, highlighting the need for additional research on sources of these contaminants in livestock dominated areas.