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. 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. We found that iris colour and activity rhythm have more likely evolved in concert than independently, and a non‐significant trend of dark eyes to evolve more easily in owl species presenting strictly nocturnal habits than in diurnal species. The transition from diurnality to nocturnality was a previous requisite for the evolution of dark irises in owls. Taken together our results are only partly consistent with the camouflage hypothesis suggesting that dark irises in owls have primarily evolved to enhance concealment in nocturnal conditions.