Context <p>Green areas within urban matrices can serve as refuges for many species, but the long-term conservation of these semi-isolated ecosystems depends on connectivity, which varies across species due to different dispersal strategies.</p> Objectives <p>This study aimed to assess ecological connectivity across the network of protected areas in Rome, one of Europe’s greenest metropolitan regions. Specifically, we examined how different dispersal strategies influence connectivity patterns and identified key ecological corridors and stepping-stone areas that facilitate multi-species movement.</p> Methods <p>We applied an integrated multi-scenario modelling framework, combining circuit and graph theory. Connectivity was modelled under nine resistance scenarios grouped into three dispersal modes, and evaluated across multiple dispersal distances.</p> Results <p>Our results reveal strong spatial differences in connectivity, with higher permeability in the western and peripheral parts of the city, and consistently low connectivity in the central and eastern sectors. We identified key ecological corridors and stepping-stone areas that facilitate movement across species groups, emphasizing the dual role of the Tiber River as both a corridor and a barrier depending on the species’ dispersal strategy. Connectivity increased with dispersal distance across all scenarios, while shared corridors were consistently associated with riverine systems and urban green spaces.</p> Conclusions <p>By identifying shared corridors and high-priority nodes under alternative dispersal scenarios, our findings provide a comprehensive assessment of connectivity in an urban landscape, capturing the diversity of dispersal strategies and landscape responses across a wide array of organisms. The proposed framework offers a transferable approach for exploring connectivity hypotheses in other urban contexts under clearly defined assumptions.</p>