Anthropogenic alterations to river systems, such as irrigation and hydroelectric development, can negatively affect fish populations by reducing survival when fish are routed through potentially dangerous locations. Non-physical barriers using behavioural stimuli are one means of guiding fish away from such locations without obstructing water flow. In the Sacramento–San Joaquin River Delta, we evaluated a bio-acoustic fish fence (BAFF) composed of strobe lights, sound and a bubble curtain, which was intended to divert juvenile Chinook salmon (Oncorhynchus tshawytscha) away from Georgiana Slough, a low-survival migration route that branches off the Sacramento River. To quantify fish response to the BAFF, we estimated individual entrainment probabilities from two-dimensional movement paths of juvenile salmon implanted with acoustic transmitters. Overall, 7.7% of the fish were entrained into Georgiana Slough when the BAFF was on, and 22.3% were entrained when the BAFF was off, but a number of other factors influenced the performance of the BAFF. The effectiveness of the BAFF declined with increasing river discharge, likely because increased water velocities reduced the ability of fish to avoid being swept across the BAFF into Georgiana Slough. The BAFF reduced entrainment probability by up to 40 percentage points near the critical streakline, which defined the streamwise division of flow vectors entering each channel. However, the effect of the BAFF declined moving in either direction away from the critical streakline. Our study shows how fish behaviour and the environment interacted to influence the performance of a non-physical behavioural barrier in an applied setting.