Disturbance by tourists of bats in their day roosts represents a potential threat to the conservation of these mammals. We assessed the effect of experimental tourist visits on behavior of the Malagasy endemic Rousettus madagascariensis (Chiroptera: Pteropodidae) in the Ankarana National Park, northern Madagascar. We measured bat behavior, including time activity budgets, under two levels of experimental human visit proximity (far and near) and illumination (low and high). These visits caused an increase in bat flights and an increase in alertness in a frequently-visited colony with faint ambient daylight. The greatest response occurred for human approaches to 5–6 m that directly illuminated the bats and the least response occurred for approaches to 12–14 m that did not directly illuminate the bats. In an infrequently-visited colony with no ambient daylight, visits that remained 12–14 m away and did not illuminate the bats directly caused an increase in alert behavior and a decrease in bat grooming behavior. The difference in response between the colonies suggests that R. madagascariensis may demonstrate an attenuated response to some frequent human visits. Alternatively, colonies roosting with some ambient light may be less sensitive to disturbance from visits, and colonies with previous experience primarily with hunting visits may be more responsive to disturbance. Maintaining a minimum visit distance of 12 m and not illuminating the bats directly, as well as not opening other roost sites to tourism, is likely to help to limit disturbance of R. madagascariensis by tourists at Ankarana.