The deep sea covers about 79% of the Mediterranean basin, including habitats potentially able to deliver multiple ecosystem services and numerous resources of high economic value. Thus, the deep Mediterranean Sea represents an important frontier for marine resources exploitation, which is embedded within the European Blue Growth Strategy goals and agendas. The deep sea is crucial for the ecological functioning of the entire basin. For this reason, the deep Mediterranean deserves protection from the potential cumulative impacts derived from existent and developing human activities. Marine Spatial Planning (MSP) has been identified as key instrument for spatially allocating maritime uses in the sea space avoiding spatial conflicts between activities, and between activities and the environment. Indeed, MSP incorporates the ecosystem-based approach (EB-MSP) to balance both socio-economic and environmental objectives, in line with the Maritime Spatial Planning Directive and the Marine Strategy Framework Directive. Despite MSP is under implementation in Europe, the Directive is not applied yet for the managing and monitoring of the environmental status of the deep sea. In the Mediterranean, deep areas fall both in internal and territorial waters, and in High Seas, and its management framework turns out to be complicated. Moreover, a certain level of cumulative impacts in the deep Mediterranean has been already identified and likely underestimated because of paucity of knowledge related with deep-sea ecosystems. Thus, the implementation of scientific knowledge and the establishment of a sustainable management regime of deep-sea resources and space are urgent. This study aims at reflecting on the best available ecological knowledge on the deep Mediterranean to incorporate conservation objectives in EB-MSP. We propose a framework to include key ecological principles in the relevant phases of any EB-MSP processes taking in consideration existing socio-economic and conservation scenarios in the region. We add the uncertainty principle to reflect on the still unexplored and missing knowledge related to the deep Mediterranean. Here, we resume some guidelines to overcome limits and bottlenecks while ensuring protection of deep-sea ecosystems and resources in the Mediterranean Sea.