The loss of Ecosystem Services (ES) associated with the direct and indirect impacts of infrastructure construction are poorly integrated into Environmental Impact Assessment (EIA) studies. In this paper, we attempt to broaden the scope of infrastructure project evaluation by incorporating ES loss as a new criterion for decision-making. This allows a systematic consideration and thus a more efficient control of natural capital loss. We distinguish between two types of ES loss: direct ES loss induced by the ecosystem conversion and indirect ES loss associated with impacts that go beyond the width of the infrastructure because of a more widespread effect on ecological functions. To illustrate the use of this additional criterion we apply our framework in two areas of interest in a high-speed rail project in France. We focus on the provision of six regulation ES: local climate regulation, global climate regulation, air quality regulation, flood protection, water flow regulation, and biological control. We study the different proposed route options to provide an example of how choices can be improved by a spatial assessment of ES loss in terms of economic loss. We show that the ES assessment may provide a stronger argument to support environmental impact assessment studies.