The data released are associated with an examination of eDNA from round goby fish (Neogobius melanostomus) in a series of field sample collections (lake nearshore and stream transport) and in vitro laboratory experiments. The round goby was used as a model for our source of eDNA in the field collections and mesocosm experiments. The field samples for lake nearshore (water and sediment) were collected from two Lake Michigan shoreline locations (Portage Lakefront, Portage, Indiana and Washington Park, Michigan City, Indiana) to examine the detectability of eDNA, the influence of sediment on eDNA estimates, and eDNA spatial and temporal resolution. The field samples for stream transport (water) were collected from Brown Ditch, a Lake Michigan stream, and were used to model and test eDNA transport. Rates for eDNA shedding and decay in water and sediment were developed using in vitro laboratory experiments and provided a comparison of eDNA behavior (persistence and degradation) and detection in water and sediment.