Adults of three amphidromous gobiid fishes, Tridentiger brevispinis, Rhinogobius similis, and Gymnogobius petschiliensis, are euryhaline and generally found in both freshwater (FW) and brackish water (BW) areas. The determining factors for their choice of habitat with different salinity have never been explored. In this study, a salinity-choice experiment was conducted using the above species captured in the FW region of the Isazu River, northern Kyoto Prefecture. For comparison, the fluvial goby Rhinogobius flumineus and BW-acclimated G. petschiliensis were also tested. We found that the three euryhaline species, including BW-acclimated G. petschiliensis, preferred BW to FW, whereas R. flumineus preferred FW. These results suggest that salinity preference did not determine habitat in these euryhaline gobiids, which were found in FW. Surveys were also conducted focusing on competitors and predators in their potential habitats. Thus, net sampling captured many other gobiid species, and an environmental DNA method detected Japanese temperate bass, a voracious predator, in the estuarine areas, suggesting that biotic factors are major determinants in the distribution of euryhaline species.