Interactions between adult individuals of the introduced Eastern mosquitofish Gambusia holbrooki and two native fish species to the Iberian Peninsula, the Iberian toothcarp Aphanius iberus and the Valencia toothcarp Valencia hispanica, were studied in mesocosm and laboratory experiments. Eastern mosquitofish always excluded both Valencia and Iberian toothcarp when the ratio introduced-to-native was at unity or favourable to the non-native species. Food availability did not decrease significantly in the mesocosm experimental units. However, specimens of native species had a greater number of empty guts than those of Eastern mosquitofish at the end of the mesocosm experiment. Ethograms were constructed based on qualitative observations in aquaria, with a special emphasis on social behaviours, in particular agonistic (which ultimately were not observed between the species). Satiety (maximum prey number) and voracity (number of prey consumed per unit of time) of the three species were measured in aquaria. The Eastern mosquitofish achieved the highest foraging values (maximum prey = 11, at 11–12 prey min−1), whereas Valencia toothcarp achieved the lowest values (maximum prey = 7, at 5–6 prey min−1). The observed interactions between Eastern mosquitofish and the two native species are discussed.