Many species of tilapia (a collection of fish species from the cichlidae family) have biological traits that make them successful invaders. Widespread herbivory has been observed across tilapia species, however knowledge on habitat preferences and tilapia interactions with native fish food webs is limited. This study was conducted three years after spotted (Pelmatolapia mariae) and Mozambique (Oreochromis mossambicus) tilapia were detected in the Mitchell River catchment, northern Australia. Tilapia diet and habitat preferences and their trophic position in native fish food webs was determined using a combination of stomach content, stable isotope, and habitat assessment analyses. Plant material was the most abundant item in tilapia stomachs, and some stomachs also contained other items in small quantities, such as fish eggs, small shrimp, and bivalves. Some niche overlap occurred between P. mariae and native omnivore or herbivore fish species. Macrohabitat type (in-channel (no tilapia), floodplain (no tilapia), weir (tilapia present)) was related to native fish food web structure, with weir food webs showing similarities to floodplain food webs. Floodplain habitats including creeks and wetlands were also most similar to weir habitats and may therefore be most at risk of colonization as tilapia spread throughout the catchment. This study clearly demonstrated that the successful rapid invasion of these two tilapia species is linked to suitable habitat with abundant food that fuels growth and reproduction. These findings increase understanding of diet related habitat use during new tilapia invasions and trophic interactions with native species.