Hydroelectric dams have threatened the conservation of migratory fish species by blocking the migration routes, and fishways are the most common mitigation strategy. We evaluated the effectiveness of the fish ladder of Porto Primavera Dam in the severely regulated Upper Paraná River, for two Neotropical migratory fish species. Overall, 563 fish (447 Megaleporinus obtusidens and 116 M. piavussu) were PIT-tagged and monitored continuously for 4 years with the RFID system. The fish were released at eight places at upstream and downstream areas and on the left and right banks: DLN (downstream-left near the ladder; < 100 m), DLF (downstream-left far; 1.200 m), DRN (downstream-right near; 1100 m), DRF (downstream-right far; 2.100 m) ULN (upstream-left near; < 100 m), ULF (upstream-left far; 1.400 m), URN (upstream-right near, 1.200 m) and URF (upstream-right far; 2.400 m). The fish performed two-way movements through the fish ladder, ascending and descending, as well as some individuals realized both movements throughout the monitoring. Entry proportion ranged from 0 to 38.5%, and was highest for the downstream release site on the left bank, nearest the ladder. Entry times ranged from 1.5 to 449.9 days. Passage percentages were 80% for fish release downstream and 100% for fish released upstream. The upstream and downstream median transit time was 0.86 and 0.93 hours, respectively and was not significant (Log-rank test, x² = 0.053; p = 0.817). Although our study evidenced that entry was poor everywhere and nonexistent from the right bank, performance of ascending and descending passage of Megaleporinus spp. through the fish ladder of Porto Primavera was high. If guidance entry conditions can be improved this fishway design holds promise as an effective solution, although additional structures may be required to achieve this.