The interplay of abiotic factors and competition has a long history in ecology, although there are very few studies on the interaction of salinity and competition in fish. Mosquitofish (Gambusia holbrooki) are among the most invasive fish worldwide, with well documented ecological impacts on several taxa such as amphibians and small native fish. It has been previously hypothesized, based on field observations, that salinity limits the invasive success of mosquitofish and provides a competitive refuge for Mediterranean cyprinodonts. We experimentally tested this hypothesis by examining the agonistic behaviour and food competition between mosquitofish and an endangered native cyprinodont (Aphanius fasciatus) at three salinities (0, 15, 25 per thousand). Intraspecific aggressive behaviour for both species was not significantly affected by salinity. As salinity increased, mosquitofish decreased their aggressive behaviour towards cyprinodonts and captured less prey. In contrast, the cyprinodonts did not change their behaviour with different salinity treatments, with the possible exception of increased defensive acts in higher salinities, but captured more prey with increasing salinity because of the reduced efficiency of mosquitofish. Our study confirms previous field observations that salinity limits the invasive success of mosquitofish and provides one of the few experimental demonstrations that it may mediate behavioural and competitive interactions between fish species. Condition-specific competition of mosquitofish might be expected with other species and ecosystems worldwide and illustrates the importance of integrating biotic and abiotic factors in the study of interspecific interactions.