Abstract: Several types of fish passage are increasingly being installed in river barriers to restore the longitudinal connectivity of fragmented riverine habitats. A vertical slot fishway installed in the Açude-Ponte weir at Coimbra (River Mondego, Portugal) was monitored to model the efficiency of this infrastructure for anadromous fishes (i.e., sea lamprey Petromyzon marinus L.; allis shad Alosa alosa L. and twaite shad Alosa fallax Lacépède, 1800) throughout 2013 and 2014 spawning seasons. Fish passage was assessed using a video recording system. Simultaneously, eight abiotic parameters, considered as potential predictors of fishway’s use by anadromous fishes, were continuously recorded during the entire study period. Boosted Regression Trees (BRTs) analysis was applied to relate the use of the fishway by sea lamprey and Alosa spp. with the abiotic predictors. To complement this data, a passive integrated transponder (PIT) antenna system was used to quantify passage efficiency for sea lampreys. Size frequency distribution and total biomass of fishes that successfully transposed the fishway was also estimated. Video data show that in the migratory seasons of 2013 and 2014, 8333 and 21979 sea lampreys passed through fishway, respectively, and a total of 7503 and 3404 Alosa spp. individuals transposed this infrastructure during the same period. About 30% of PIT tagged lampreys transposed the fishway. BRT models identified the river flow as the most important variable to explain use of the fishway by anadromous fishes. The relationships of this variable with the fish passages suggest that sea lampreys and Alosa spp. use the fishway preferentially at relatively low flow conditions that maximize the attraction efficiency of the fishway entrance. Finally, results obtained with this study were used to implement a sub-sampling methodology (video processing) to estimate the total amount of fish (lamprey and shads) that annually use the fishway during their spawning migrations.