Dumped munition in the German North Sea and Baltic Sea pose environmental risks as corrosion of the munition shells results in the leakage of the explosive 2,4,6-trinitroluene (TNT) into the marine environment. Uptake of TNT by marine biota and the associated negative effects on organisms are of major concern. This dataset reports behavioral responses of three-spined stickleback (Gasterosteus aculeatus) to environmentally relevant concentrations of TNT. Experimental sticklebacks were laboratory-bred and held in groups of 30 individuals in 60 L tanks in the fish facilities at the Thünen Institute of Fisheries Ecology in Bremerhaven. Parental sticklebacks originated from the Weser estuary (Luneplate, Bremerhaven, Germany, 53°28'36.9" N; 8°31'08.9" E) and were collected in April 2023. A total of 60 sticklebacks were tested in a controlled laboratory setup at the fish facilities at the Thünen Institute in Bremerhaven, containing two hideout zones formed by artificial plants. Each hideout was connected to an infusion system delivering either a TNT solution (100 µg/L) or control water into the zone currently occupied by the fish. Experimental trials were video-recorded to enable post hoc behavioral analysis. Behavioral metrics included the total time spent in the exposed hideout zone (s), latency to first leave the exposed hideout zone (s), and the number of crossings between hideout zones.