Pumpkinseed Lepomis gibbosus (L.) are successful invaders in Europe, where this species exerts multiple ecological effects, mainly through trophic interactions. Behavioural interference represents a potential impact for native fauna and this is of particular conservation concern in the Iberian Peninsula because of the highly valuable endemic fauna inhabiting streams of this region. However, aggressive interactions have not previously been examined under natural conditions in Iberian fresh waters. To address this gap in knowledge, the aim of the present study was to assess the effect of pumpkinseed aggression on endemic fauna of an Iberian stream, the River Bullaque (central Spain). In September 2009, we analysed the aggression and environmental contexts of these behavioural interactions by snorkelling: aggressor size, aggression type, shoal size, previous activity to aggression, recipient species, response to aggression, microhabitat structure and prey availability. Small pumpkinseed displayed more threat and fewer pursuit behaviours relative to medium and large individuals, reflecting an ontogenetic behavioural shift from low to high aggression intensity. Small aggressors came from large shoals, with bottom feeding being the most frequently observed activity prior to an aggressive interaction; whereas large pumpkinseed were less gregarious and they were mostly ambulating within the water column prior to aggression. Recipient species of aggression included non-native crayfish and fishes, and more importantly, endemic fishes and frogs. Retreat was the most common response to aggression, irrespective of aggressor size. Small pumpkinseed displayed aggressive behaviours over coarse substrata containing elevated macrobenthos biomass; whereas aggression by large individuals was observed in deeper waters. These findings suggest that small and large pumpkinseed exert a high impact on other stream residents through aggression in competition for food and territory defence, respectively. This study highlights the usefulness of direct observations in the wild for assessing the effects of behavioural interference of invasive fishes on Iberian aquatic communities.