Received October 13, 2021; revised April 22, 2022; accepted August 15, 2022Abstract—In order to understand the success of the invasion of a non-native fish, it is important to determine its fundamental competition characteristics such as its tolerance to habitat differences and feeding plasticity. This study aimed to determine the invasive characteristics of Gambusia holbrooki Girard, 1859 by investigating the diet and feeding ecology of this non-native fish inhabited in lotic and lentic habitats. A total of 164 G. halbrooki specimens (SL 1.4–4.3 cm) were investigated from the lotic habitats, while 101 (SL 1.6–3.3 cm) ones were from the lentic habitats. The food spectrum of the fish in lotic habitats was composed of 11 food items predominantly of the Diptera (80.9, MI%) and Plant (11.8, MI%). In lentic habitats, the species consumed 10 different food types, and Diptera (96.0, MI%) was followed by the low values of zooplankton groups (Cladocera; 2.2, MI% and Copepoda; 0.8, MI%). The fish that inhabited both lotic and lentic habitats have shown a generalist feeding strategy; while the importance of each food item in the diet indicated the generalization, Diptera presented with high values was the evidence of a relatively narrow niche of the fish in both habitats. An ontogenetic shift was observed in the diet of G. holbrooki and while the diet of juveniles was more limited, the diversity and sizes of food items consumed have increased in parallel with the increase in the size of the fish. With these results, it was thought that G. holbrooki could show a food competition with native fish species in the region by feeding mainly on Diptera, which is also an important food source for most of the native fishes and their young individuals.