Artículo de publicación ISIMediterranean rivers are characterised by strong environmental constrains and species-poor, highly endemic fish fauna. In Europe, these systems are exposed to multiple stressors due to extensive human activities. Studies on the effects of some stressors on riverine fish are available but complex responses of fish assemblages to interplay of flow alteration with physical habitat changes and invasive species have not been evaluated up to date. This study analysed the response of functional diversity of fish assemblages to multiple stressors in the Segura River system in the southern Spain. Fish assemblages were sampled in 16 sites in two consecutive periods (2009–2010 and 2013–2015). Subsequently, we assessed the responses of functional specialisation, originality and entropy (based on nine functional traits and abundances) as well as species richness and abundance to interplay of flow regime alteration and ecological status, fragmentation as well as non-native species abundance across spatial and temporal scales. The governing role of flow regime in structuring fish assemblage was superimposed on physical habitat changes, water quality deterioration and fragmentation as well as the presence of non-native fish species. We found an increase of species richness and abundance but decrease of functional specialisation and originality in river reaches with high level of base flow and more stable hydrological conditions. Opposite pattern was observed in reaches with severe reduction of base flow and marked inversion in the seasonal pattern of high and low flows. We postulate that the use of tools that consider the functional identity of the species as method to assess the effects of environmental alterations on fish biodiversity could improve conservation measures for Mediterranean fish fauna. Furthermore, design flows that mimic natural flow regime patterns characteristic for Mediterranean rivers are a promising tool to provide environmental conditions that would favour native fish within the assemblage and benefit their conservation.