Microbial antibiotic resistance is a naturally occurring phenomenon that has grown in part with the use of antibiotics in agricultural operation. There are also known connections between genes for metal homeostasis and antibiotic resistance, and either antibiotics or metals may select for both kinds of genes. Antibiotics, metals, and their associated genes have the potential to enter agricultural drainage channels and migrate to downstream locations through receiving water bodies. A relatively new agricultural best management practice—the two-stage channel design—functions by sequestering sediments and dissolved constituents as they flow through agricultural ditches from surface runoff and tile drainage discharge. Sedimentation in agricultural watersheds may entrap aggregate pollutants including antibiotics, metals, and associated resistance genes before transport into the drainage system. Here, we characterized the abundance and diversity of 22 antibiotic resistance and metal homeostasis genes in three two-stage channels that had self-developed in an area dominated by agricultural land use. Additionally, we analyzed the sediments for 17 antibiotics and nine metals that could affect the selection of these genes. In these rural systems that drain into Lake Erie, the abundance of antibiotic resistance and metal homeostasis genes were on the lower end of ranges (e.g., <106 gene copies g−1 of intI1) reported in other riverine and lake systems, with only five genes—intI1, aacA, mexB, cadA, and merA—differing significantly between sites. The diversity of antibiotic resistance and metal homeostasis genes for these sediment samples were largely similar to other human impacted environments. Few antibiotics were detected in two stage channel sediments, with concentrations below the quantifiable limits (<0.02–34.5 µg kg−1 soil) in most cases. Likewise, metals were present at what could be considered background concentrations. Despite serving as important drainage channel features in a region dominated by agricultural land use, results serve as an important baseline reference against which other monitoring studies can be compared to assess the perturbation of antibiotics and metals on agricultural channel sediments.