Data included are from a series of field sample collections from Lakes Michigan and Huron, and laboratory mesocosms targeting the round goby fish (Neogobius melanostomus). The round goby is a benthic fish that has heavily invaded four of the five Laurentian Great Lakes. Because it inhabits a variety of substrates, including coastal breakwaters, traditional methods (e.g., trawling, trapping) are inadequate to quantify overall population size. Environmental DNA (eDNA) may be a viable option for improving detection and quantification of the species. Field data include number of round goby caught and associated ambient conditions of the aquatic matrix (temperature, pH, turbidity, conductivity, dissolved oxygen). Mesocosm data include information about mesocosm test (DNA shedding, decay), information about the individual fish used (length, weight), ambient conditions of the aqueous matrix (pH, turbidity). Data from laboratory testing of both field and mesocosm water include results from quantitative polymerase chain reaction (qPCR) method for detection of round goby-specific DNA (DNA quality, quantity). Data are being used to correlate round goby individuals with eDNA present in the water and to calculate rates of DNA shedding by the round goby and rates of DNA decay.