Tourism represents a key economic sector worldwide, constituting great leverage for local economic development but also putting noticeable environmental pressures on local natural resources. Ecotourism may be a viable alternative to mass tourism to minimize impacts on ecosystems, but it needs shared sustainability standards and monitoring tools to evaluate impacts. This paper presents a first methodological proposition to calculate the environmental impact of ecotourism packages through the use of an ad-hoc, customized version of the Ecological Footprint methodology. It follows a participatory, bottom-up approach to collecting input data for the four main services (Accommodation, Food & Drinks, Activity & Service, and Mobility & Transfer) provided to tourists through the use of surveys and stakeholders engagement. The outcome of our approach materializes in an excel-based ecotourism workbook capable of processing input data collected through surveys and returning Ecological Footprint values for specific ecotourism packages. Although applied to ecotourism in Mediterranean Protected Areas within the context of the DestiMED project, we believe that the methodology and approach presented here can constitute a blueprint and a benchmark for future studies dealing with the impact of ecotourism packages.