Substrate and depth are known to be crucial factors in determining the spatial distribution of benthic communities. Herein, we explored if the nature of the substrate, living (i.e., macroalgae) and non-living (i.e., bare rock), may influence the hydroid assemblages of the Tuscany Archipelago (Italy), in relation to the bathymetric gradient. Samples were collected in June 2019 and 2020 in two islands, Montecristo and Giglio. Overall, 16 families (12 Leptothecata and 4 Anthoathecata) and 74 species were identified, with exclusive species observed for each substrate type and depth. The pattern of assemblages’ distribution indicates an interaction between two factors (substrate and depth) also depending on the considered island. The DISTLM-forward analysis revealed that the percentage coverage of several algal taxa significantly explained a part of the observed variation in the hydroid assemblages. This study improves the knowledge of the zoobenthic communities of the Tuscan Archipelago National Park and points out the important role of the living substrate and its variation in the distribution patterns of the hydroids assemblages. In addition, following the Italian legislation and the EU Biodiversity Strategy for 2030, this paper contributes to improve the knowledge of the biodiversity of the Tuscan Archipelago providing a baseline for the biodiversity management of the forthcoming marine protected area.