In promoting biodiversity conservation and ecosystem service capacity, landscape connectivity is considered a critical feature to counteract the negative effects of fragmentation. Under a Green Infrastructure (GI) perspective, this is especially true in rural and peri-urban areas where a high degree of connectivity may be associated with the enhancement of agriculture multifunctionality and sustainability. With respect to GI planning and connectivity assessment, the role of dispersal traits of tree species is gaining increasing attention. However, little evidence is available on how to select plant species to be primarily favored, as well as on the role of landscape heterogeneity and habitat quality in driving the dispersal success. The present work is aimed at suggesting a methodological approach for addressing these knowledge gaps, at fine scales and for peri-urban agricultural landscapes, by means of a case study in the Metropolitan City of Rome. The study area was stratified into Environmental Units, each supporting a unique type of Potential Natural Vegetation (PNV), and a multi-step procedure was designed for setting priorities aimed at enhancing connectivity. First, GI components were defined based on the selection of the target species to be supported, on a fine scale land cover mapping and on the assessment of land cover type naturalness. Second, the study area was characterized by a Morphological Spatial Pattern Analysis (MSPA) and connectivity was assessed by Number of Components (NC) and functional connectivity metrics. Third, conservation and restoration measures have been prioritized and statistically validated. Notwithstanding the recognized limits, the approach proved to be functional in the considered context and at the adopted level of detail. Therefore, it could give useful methodological hints for the requalification of transitional urban–rural areas and for the achievement of related sustainable development goals in metropolitan regions.