Birds, due to their multiple colourful displays, constitute a classic paradigm for the study of colour evolution. Although avian eyes are remarkably coloured, the functional basis behind inter‐specific variability in iris colouration remains poorly understood. Owls are an ideal system to shed light on the role of ecology in promoting iris colour evolution as they show inter‐specific variation in iris colour and in niche specialization with some species being strictly nocturnal and others active during the day. Owls perching for hunting at night might be unnoticed by both predators and their prey if they had dark irises, which would predict that dark irises were more likely to evolve in strictly nocturnal species than in diurnal ones. Using phylogenetic comparative models, we tested the camouflage hypothesis for eye colour. The proportion of dark‐eyed owl species is higher among strictly nocturnal owls than among diurnal ones. Ancestral state reconstruction revealed that the owl ancestor of the family Strigidae was more likely bright‐irided whereas the ancestor of the family Tytonidae was more likely dark‐irided. Our results show robust support for the coevolution of iris coloration and nocturnality in the owls, and suggest that shifting to a nocturnal niche would be a prerequisite leading to the evolution of dark eyes in owls. The specific evolutionary pathway by which iris coloration and activity rhythm coevolve, however, remains to be investigated further as we have found only partial support for the idea that dark irises in birds might be an adaptive feature evolved due to the selective advantage of concealment from undesired visual receptors.