Hoplosternum littorale is a South American catfish (Family Callichthyidae) first discovered in the United States in 1995 in Florida. The presence of H. littorale was documented from early 2002 to late 2003 in Lake Tohopekaliga (Kissimmee River Basin) in central Florida. In this paper, 22 H. littorale nests and nest sites are described. The characteristic bubble nests were present from late May to early September, with number of nests peaking in August when water stage and temperature were both high. Nest habitats (shallow, open marshes) and timing of nest construction (rainy season) were similar to what has been reported for H. littorale in its native range. Most nests (n = 14) were in areas dominated by Hydrilla verticillata and constructed largely from parts of this Asian aquatic plant, representing a unique association between two non-native species. Nevertheless, during August, as water levels increased, nesting shifted from H. verticillata-dominated communities to use of inshore grass zones dominated by Luziola fluitans. Knowledge of H. littorale nesting seasonality and habitat preferences may be useful for any efforts to control or manage this invasive fish.