Land-use change poses a significant threat to biodiversity, particularly in insular ecosystems where species exhibit higher levels of specialisation. Despite the importance of tropical islands for biodiversity conservation, the impacts of land-use change on the diel activity of insular species remain poorly understood. We addressed this gap by investigating the diel activity patterns of insectivorous bats in response to land-use change in São Tomé Island, Central West Africa. Using passive acoustic detectors, we surveyed insectivorous bats across forests, shaded plantations, oil palm plantations, agricultural and urban areas. We examined (1) how intraspecific diel activity varies between forest and human-altered land-use types, and (2) how interspecific diel activity varies within the same land-use type, by quantifying overlap coefficients in species activity. Based on 19,383 bat passes from four species, we found that the degree of overlap in species-specific activity between forests and human-altered land-use types varied between species, with the least overlap being observed between forests and urban areas for Miniopterus newtoni, and between forests and oil palm plantations for Chaerephon spp.. Overlap in the activity of different species was lower within less or not disturbed land-use types (forests and shaded plantations), where more species were present. Overall, diel activity of insectivorous bats altered according to the magnitude of structural land-use change. Such bat responses might be eventually due to the different temporal prey availability and interspecific competition in the altered land-use types. These findings comprise baseline knowledge informing evidence-based management actions across tropical island systems.