The introduction of Xylella fastidiosa subsp. pauca in Puglia (Italy) has disturbed the flow of ecosystem services (ES). We assessed the short- and long term impacts of this disease and the control measures against it on ES provisioning and biodiversity in the olive-producing agroecosystem of Puglia at a landscape level by using an ES framework in combination with expert knowledge elicitation. Experts estimated the minimum, maximum and most likely relative reductions in ES delivery and biodiversity components in the affected area. The estimates were used to fit a PERT distribution. Results show that the loss of olive trees (the main woody plant in the region) in general, and of centenarian olive trees in particular, has affected ES delivery. The delivery of ES is estimated to diminish by 34% and 30% in the short- and long term, respectively, mainly due to the impact of the disease on food (olive) production, ornamental resources, soil erosion regulation, primary production and cultural heritage. Biodiversity components are estimated to diminish by 28%, mainly due to the impact of the disease on genetic diversity and habitats of high conservation value. The study provides a first assessment of the wider environmental impacts of Xylella fastdiosa subsp. pauca, which can serve as a basis for formulating hypotheses for further investigation.