This study investigates the impact of tourist activities on penguin behaviour on Barrientos Island, an intensively visited site in the Antarctic Peninsula. By simulating tourist presence and activities, we assessed the behavioural responses of two species, Pygoscelis antarcticus and Pygoscelis papua, and their differences with regard to two specific factors: nest position (perimetral/non-perimetral) and nesting site size (large/small). The findings reveal that both species were sensitive to human presence, especially when visitors talked while approaching the nesting sites. We observed greater behavioural differences regarding the position within the colony for gentoo penguins (P. papua). For this species, the size of the nesting sites was also an important factor affecting its response to human presence, with the large nesting sites being less affected. This trend was not shared by chinstrap penguins (P. antarcticus), whose behaviour towards visitors was not affected significantly by either factor. We recommend mitigating tourists’ impact by maintaining their viewing distance at greater than 10 m and by having them move quietly and slowly around these penguin species.