New types of fish guidance structures with vertical curved bars and a subsequent bypass system represent a promising technical solution for the protection and guidance of downstream moving fish at run-of-river hydropower plants and water intakes. These so-called "curved-bar rack bypass systems" (CBR-BSs) function as a mechanical behavioral barrier and are characterized by low hydraulic losses, a symmetrical downstream flow field and an overall high fish guidance efficiency in the laboratory for a wide array of European freshwater fish species. This paper presents the results of the hydraulic and live-fish laboratory tests of an optimized CBR-BS configuration with a bar spacing of 50 mm and 30° rack angle to the flow direction. The tests were conducted with six different fish species in an ethohydraulic laboratory flume at different approach flows (0.5 m/s, 0.7 m/s) and different bypass entrance velocities (0.6-1.0 m/s). A numerical model was used to simulate the flow fields in the CBR-BS in order to link the fish behavior to the hydrodynamic cues created by the CBR-BS. Lower approach flow velocities decreased the hydraulic cues of the CBR, which led to more rack passages. A 20% velocity increase towards the bypass entrance significantly increased the fish guidance efficiency compared to a 40% velocity increase. The tested CBR-BS resulted in overall higher interspecies fish protection and guidance efficiencies compared to the more commonly applied horizontal-bar rack with a narrow bar spacing of 20 mm. Recommendations for a sustainable and cost-effective application of CBR-BSs are given.