Conservation efforts are increasingly supported by ecosystem service assessments. These assessments depend on complex multi-disciplinary methods, and rely on a number of assumptions which reduce complexity. If assumptions are ambiguous or inadequate, misconceptions and misinterpretations may arise when interpreting results of assessments. An interdisciplinary understanding of assumptions in ecosystem service science is needed to provide consistent conservation recommendations. Here, we synthesise and elaborate on 12 prevalent types of assumptions in ecosystem service assessments. These comprise conceptual and ethical foundations of the ecosystem service concept, assumptions on data collection, indication, mapping, and modelling, on socio-economic valuation and value aggregation, as well as about using assessment results for decision-making. We recommend future assessments to increase transparency about assumptions, and to test and validate them and their potential consequences on assessment reliability. This will support the taking up of assessment results in conservation science, policy and practice.