Multiple studies have shown that human disturbance can have negative impacts on wild penguin populations. Penguins in zoos may also be susceptible to negative impacts from humans, but this has not previously been investigated. We examined the visitor effect on a group of 25 little penguins, Eudyptula minor, by randomly imposing two treatments: (1) no visitor contact, which was achieved by closing the penguin exhibit on study days and (2) exposure to visitors, with the penguin exhibit open as usual. Treatments were imposed for 1-day periods, with five replicates of each treatment (total of 10 study days). Instantaneous point sampling and continuous sampling were used to record penguin behaviour including proximity to visitor viewing area, surface swimming, diving, vigilance, visibility, resting and intra-group aggression during a total of 3 h on each of the 10 study days. When exposed to visitors, penguins showed increased levels of aggression (P = 0.02), huddling (P = 0.049) and behaviours indicative of avoidance of visitors including increased time spent positioned behind enclosure features (P = 0.024) and increased distance from the visitor viewing area (P = 0.002). These behavioural results suggest that the presence of visitors or some aspect of visitor behaviour may have been fear-provoking for these penguins. To generalize beyond this group of animals and this enclosure requires further research.