Urbanisation is a rapidly growing global phenomenon leading to habitat destruction, fragmentation and degradation. However, urban areas can offer opportunities for conservation, particularly through the presence of green spaces which can even provide important habitats for imperilled species. Spiders, which play crucial roles in ecosystem functioning, include many species that can successfully exploit urban environments. Placed in the middle of the Mediterranean global biodiversity hotspot, Italy possesses an exceptionally rich spider fauna, yet comprehensive data on urban spider communities are still limited. More information on urban spiders in Italy would be extremely beneficial to support conservation efforts, especially in central and southern Italy, where knowledge on the spider fauna is largely incomplete. The current study focused on the spider diversity of a large protected area (Appia Antica Regional Park) in urban Rome, Italy. A total of 120 spider species belonging to 83 genera and 28 families were identified, with 70 species being new records to the Province of Rome, 39 to the Latium Region and two (Pelecopsis digitulus Bosmans & Abrous, 1992 and Palliduphantes arenicola (Denis, 1964)) to Italy. Forty-one species were recorded during autumn/winter sampling and 107 in spring/summer. The spider fauna recorded from the study area included about 37% of the total spider fauna known from the Province of Rome, 28% of that of the Latium Region and 7% of the entire Italian territory. The most represented families in terms of species richness were Gnaphosidae and Linyphiidae, which accounted for more than 40% of the sampled fauna. Lycosidae were the most abundant family (29% of captured individuals), followed by Zodariidae (16% of captured individuals), Linyphiidae (13% of captured individuals) and Gnaphosidae (7.5% of captured individuals). From a biogeographical point of view, most of the collected species belonged to chorotypes that extend for large areas across Europe and the Mediterranean. The research highlights the role of urban green spaces as refuges for spiders and the importance of arachnological research in urban areas as sources of information on spider biodiversity at larger scales.