Invasive species have significantly impacted freshwater ecosystems in recent decades. The eastern mosquitofish Gambusia holbrooki Girard, 1859, the chameleon fish Australoheros facetus (Jenyns, 1842), and the sewage snail Physa acuta Draparnaud, 1805, have invaded numerous countries worldwide, including Chile. To date, these species have not been found in syntopy within this country. Through morphological and molecular analyses, we achieved precise taxonomic identification of these three species in Lake Santa Elena, & Ntilde;uble Region, thereby filling the gap in their invasive ranges. Given the high potential for the spread of these invasive species, we recommend conducting additional surveys in other water bodies in Chile where they have not been previously recorded. Considering that there are rare cases successful in eradicating invasive species, priority should be given to protecting non-invaded water bodies from new invasions though it is also important to make persistent efforts for conservation of native communities in the invaded ecosystems.