Hydropower dams come with high ecological and social costs, not least concerning longitudinal connectivity in rivers, which causes declines and sometimes local extinctions of fish species. Fishways are widely used to allow fish to pass dams, but their efficiency is highly variable between species and sites. Many species, and at places entire fish communities, remain understudied, likely hindering the implementation of effective remedial measures. Here, we studied fish passage behaviour in a vertical slot fishway in the Po River, Italy. Almost 1000 individual fish of nine species, representing the local fish community, were tagged and released within and downstream of the fishway. The only species passing the fishway at relatively high numbers were potamodromous barbel and Italian chub, and for these species passage success was positively related to fish size. Passage was more likely to occur at night than during the day for barbel, but not for chub. In relation to the dispersal of invasive species, it is noteworthy that a few individuals of wels catfish and common carp passed the fishway.