Biological invasions are widely regarded as the second major cause of the current global biodiversity crisis. Freshwater environments in particular include many habitats and species of conservation concern. Among those, amphibians are threatened worldwide by habitat loss and by the introduction of alien taxa, including pathogens and fish. The North American mosquitofish Gambusia spp. have been introduced throughout the world for the biological control of mosquitoes, and have established alien populations in over 110 countries. Their impact on native freshwater vertebrates, especially in Europe, is still poorly known. Here, the predatory impact of mosquitofish on the larval stages of four European amphibian species ( Hyla intermedia , Triturus carnifex , Pelophylax kl. hispanicus , and Bufotes balearicus ), locally abundant in Central Italy, has been investigated under laboratory conditions to assess whether any difference occurs in species vulnerability and interactions with the predator, as their breeding sites are often invaded by mosquitofish. Larval mortality and fish attacks, as well as observations on fish predatory behaviour, were recorded at four time intervals (10 min, 1 h, 6 h, and 24 h) and at three different prey abundances (with five, 10, and 20 larvae). A significant difference in predation rate for species, exposure time, and larval abundance occurred, with H. intermedia being the most vulnerable species and B. balearicus being the least vulnerable species. An increase in exposure time increased the rate of predation, whereas small groups of larvae (i.e. 〈10 individuals) appeared to be far more vulnerable to fish attack compared with the larger groups. Mosquitofish show considerable potential to prey upon the larvae of European amphibians, representing a serious threat for their conservation. Appropriate management and legislative strategies are thus necessary to prevent the further introduction and spread of this alien fish in natural environments.