Wildfires are a common phenomenon in Mediterranean regions that is becoming increasingly frequent and severe, causing several environmental concerns, of which ash runoff represents an important source of disturbance for aquatic organisms, in particular for fishes. Studies on the behavioural response of fishes to wildfire ash runoff are scarce and seldom include cyprinid species. The goal of this study was to investigate in a 3-artificial flume channel mesocosm, the behavioural and hepatosomatic condition responses of a native widespread potamodromous fish, the Iberian barbel (Luciobarbus bocagei), previously exposed for 24 h to different concentrations of wildfire ashes: 0.0 g/L (the control, no ash), 1.0 g/L (low concentration) and 2.0 g/L (high concentration). Behavioural parameters included i) routine activity, ii) boldness and iii) shoaling cohesion. The hepatosomatic index (HSI) was further determined to assess the health condition of fish. Significant differences on fish behaviour parameters were detected between the control and the high concentration of ash. Accordingly, i) an increasing proportion of fish were found on resting activity (56.2% vs 30.6% in the control), whereas the proportion of fish on searching behaviour (58.4% in the control) decreased (41.5%); ii) the proportion of bolder individuals was found to decrease (42.5% in the control vs. 29.4%) and iii) the same trend was detected for shoaling cohesion (61.3% in the control to 33.8%, of all fish within a body length of each other). Such differences were paralleled by an increase in the HSI from 1.62% (control) to 2.40% (high concentration). The present study shows that even short duration exposure to ash-loaded runoff can alter fish behaviour and hepatosomatic condition and highlights the need to maintain an unfragmented river network, or, when this is not possible, to prioritize the removal or retrofitting of barriers to increase movement dispersal and provide conditions for species recovery from fire-disturbances.