ABSTRACT Non-native species are a major driver of biodiversity loss. Aquaculture activities play a key role in introductions, including the escape of fishes from fish farm facilities. Here, the impact of flooding due to El Niño rains in 2015/2016 in the Lower and Middle Paranapanema River basin, southern Brazil, was investigated by evaluating fish escapes from 12 fish farms. The flooding resulted in the escape of approximately 1.14 million fishes into the river, encompassing 21 species and three hybrids. Non-native species were the most abundant escapees, especially Oreochromis niloticus (Linnaeus, 1758) and Coptodon rendalli (Boulenger, 1897) (96% of all fish). Only seven native fishes were in the escapee fauna, comprising 1% of all fish. Large floods, coupled with inadequate biosecurity, thus resulted in considerable inputs of non-native fish into this already invaded system.