Mobile Bay is a dynamic estuary home to a diverse faunal assemblage, which includes several species of batoid fishes (Chondrichthyes: Batoidea). To better understand the dynamics of this batoid assemblage, batoids were opportunistically sampled from 440 trawls performed in and around Mobile Bay from 2016 to 2017. The species Hypanus sabinus and Gymnura lessae were the most common batoids collected (86% of catch). PERMANOVA analysis found the variables day length, location, year, and water temperature best described catch variability. Furthermore, stomach contents from Gymnura lessae were sampled to investigate its diet. Most prey were heavily degraded, thus DNA metabarcoding was used to enhance prey identification. Most prey (88.3%) were from the families Sciaenidae and Engraulidae, and the variables season and sex best explained the dietary variability. These data will be necessary for modeling potential habitat and dietary shifts of Mobile Bay’s batoids as climate change and anthropogenic disturbances alter estuaries.