A repulsive effect, that some induced primary stimuli, like sound and light, is known to be provoked in fish behavior. In the present study, two strobe light frequencies, 350 flashes/minute and 600 flashes/minute, were tested in laboratorial conditions, using three native freshwater fish species of northern Portugal: Brown trout (Salmo trutta), Northern straight-mouth nase (Pseudochondrostoma duriense) and Iberian barbel (Luciobarbus bocagei). The results showed a differential repulsive behavior of the fish species to light stimulus, and particularly to a frequency of 600 flashes/minute. S. trutta presented the most repulsive behavior, whereas the L. bocagei showed less repulsion to the light stimulus. No relevant differences were found between pre-test and post-assessments, confirming a rapid recovery of natural fish behavior after the deterrent effect. The results highlighted the potential of behavioral barriers, particularly in salmonid streams, based on strobe light stimulus.