Tornillo Creek, a tributary of the Rio Grande in Texas, United States, has historically been an important nursery and spawning habitat for several native fish species. We examined variation in the seasonal fish assemblages in Tornillo Creek within and between two time periods (1967–1970 vs. 2009–2011), and contemporary fish assemblage–environment associations, in order to understand what environmental factors were associated with seasonal fish abundance and occupancy patterns. Our results indicated that fish assemblages were very different between the two time periods. Contemporary seasonal fish assemblage patterns in Tornillo Creek were less variable than historical assemblages and were linked to several environmental factors including water temperature, stream depth, and current velocity. We suggest that the maintenance of stream flow and connectivity are important for the immigration and emigration of certain riverine fishes in Tornillo Creek and that decreases in stream flow could accelerate the domination of the tolerant species in the creek.