Personality characteristics (boldness, sociability, aggression) of individual animals vary within and amongst species, and may mediate foraging behaviour and breeding success, and affect capacity to adapt to environmental change. This research project measured a personality character (boldness) in two species of penguins housed within a shared facility at Sea World, through a series of controlled and fully randomised experiments. A colony of 26 gentoo and 16 king penguins under human-care were presented with a series of replicated treatments consisting of familiar and novel keepers, objects, food, and a control (keeper, no object) over a 10-day period. In each experimental trial of 300 secs, latency to first approach of the novel object and/or keeper was measured; a positive boldness score occurred if a penguin was faster to approach the 1 m zone in the treatments relative to the control, the inference of this being that a penguin’s boldness is proportionate to the positive value along the shy–bold continuum. The influence of individual characteristics (sex, age, species) on ‘boldness’ was examined and the effect on penguin behaviour. This study has implications on management strategies (e.g., enrichment activities, guest encounters) to foster positive affective states and well-being of cohabitating species under human-care.