Coastal ponds are peculiar and threatened habitats, considered biodiversity hotspots. In many ecosystems ponds can be a valuable resource for the conservation of freshwater biodiversity. The Castelporziano Presidential Estate is a Mediterranean protected area hosting numerous ponds which are suffering severe droughts and a general decline in number and hydroperiod length. Hydroperiod changes can be particularly relevant for biodiversity inhabiting ponds, in particular diatom communities. Here, for the first time, we evaluate the conservation status of Mediterranean ponds inside the Castelporziano Reserve. The aims of this research are to i) investigate the influence of environmental variables on diatom communities of ponds; (ii) analyse diatom community structure in function of species richness and competitive interactions; (iii) highlight the potential and effectiveness of diatom ecological guilds in supporting the interpretation of the ecological status of threatened small waterbodies. Overall, 122 diatom taxa in the 29 ponds were described. Nitzschia (18 species) was the most species-rich genus, followed by Gomphonema (14) and Navicula (13). 17 species were included in the Red List of endangered species. Intra-pond distance and anthropogenic impacts didn't influence diatom assemblages. On the contrary, diatom communities were influenced by altitude and the distance from the sea. Furthermore, temporary ponds showed the lowest values for Shannon, evenness and Simpson indexes Considering Beta-diversity, both Bray-Curtis and Jaccard dissimilarity matrices highlighted a large dissimilarity between sampling sites. Inter-variability between sites and intra-variability within the sites resulted both very low. Our results highlight that diatom species are not influenced by hydroperiod length. Moreover, a structured diatom community emerged, where segregation processes predominating over aggregation ones. Finally, the average water quality of the ponds, calculated using the EPI-D index, was described as “good”. This study represents the most complete checklist of diatom communities inside coastal ponds of Castelporziano, providing valuable insights into the effects of natural and anthropogenic influences on ponds conservation and management activities. However, spatial-temporal changes and anthropogenic impacts should be deeply investigated.