For a long time the bacterial phylum of Chlamydiae exclusively consisted of one family of obligate intracellular bacteria, the Chlamydiaceae, which encompassed causative agents of severe diseases. In the 1990s, environmental chlamydiae were discovered as symbionts of free-living amoebae and other eukaryotic hosts. During a sampling campaign in September 2008, while monitoring Planctomycetes, we retrieved 20 almost full-length 16S rRNA gene sequences affiliated with Chlamydiales from a lake at the Tyrrhenian coast of central Italy (Lago di Paola, Latium). Two main clusters were identified. The nine sequences within the tight cluster I shared ∼98% identity, just like the six sequences of cluster II. The 16S rRNA sequence identity between the two novel groups was with 88% higher than with all known families of the order Chlamydiales. Four types of less frequent chlamydial 16S rRNA sequences were also detected. Two oligonucleotide probes were designed, and optimized. Chl282 targets the cluster I and almost all other Chlamydiales, while Chl282bis targets the cluster II and few other sequences. By catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH), we identified in the Lago di Paola picoplankton abundant tiny cells with dot-shaped morphology and, interestingly, rarely also protists with intracellular pleomorphic chlamydiae. Abundances of the novel chlamydial clusters were up to 5 × 104 cells per millilitre. The two clusters were also detected in similar numbers during a second sampling in October 2010. This confirmed the relevance of the two newly described clusters of chlamydiae in Lago di Paola, not only enlarging the knowledge on the biodiversity of environmental chlamydiae in aquatic habitats, but also raising sanitary issues that should be addressed in the future.