All over the world marine waters are under increasing pressure from human activities affecting marine ecosystems. Several EU Directives require assessment of the condition of marine environments; in particular the Marine Strategy Framework Directive (MSFD) applies an ecosystem approach to the management of human activities. In this context, the mapping of pressures in a standardized and comparable way is a critical step to assess the spatial pattern, the temporal change and the ecological impacts of human pressures. Within the MSFD, one of the stressors directly affecting the seafloor is the Physical Loss (PL) pressure, representing the permanent or long-term alteration of marine habitats. The main purpose of this study was to propose a method to estimate the spatial extent of PL pressure in the framework of the Initial Assessment phase of the MSFD. Furthermore, considering that human activities PL-related cause the loss of benthic habitats, and that the Mediterranean sea is characterized by sensitive and protected habitats such as the biogenic substrates sensu MSFD, the distribution of PL pressure was overlaid with the distribution of the seabed habitats to estimate the loss of biogenic substrates. This study represents a useful tool for establishing the baseline condition for PL pressure, to compare future conditions and to evaluate different management scenarios. Moreover, it allows identification of the areas where pressure tends to accumulate as ‘hot spots’ on which to focus in future impact analyses and the areas where few stressors are present.