Owl cafes, where customers view and interact with owls, have become popular in Japan. There are multiple aspects of the environment which may be stressful to nocturnal owls, including lighting, tethering and frequent interactions with humans but, to date, welfare has not been investigated. This preliminary study examines the effects of owl cafes and customers on the physiological stress (faecal corticosterone levels [FCL]) and behaviour of the owls. Seven eagle owls (Bubo bubo) and two African scops owls (Ptilopsis leucotis), in two cafes, were studied over an eight-day period. Cafe A (n = 5) was closed for one day per week, whilst cafe B (n = 4) was open every day. In cafe A there was higher FCL in owls during open days than closed days suggesting that the conditions on open days increase stress in owls. Eight of nine owls showed evidence of some aversion or avoidance of humans, whilst no affiliative behaviours were observed. The number of visitor interactions was not associated with the level of physiological stress; and when comparing owls, there was a tendency for individual owls with a higher percentage of aversive responses to customers to also have higher FCL. Close human interactions of a negative nature, that result in aversion, may be a significant stressor, but further research is required.