Water systems have been shown to be a potential source of Chlamydiae, intracellular symbionts of eukaryotes. However, their diversity is likely underestimated, and natural hosts remain undetermined in many cases. In this study, we combined PCR-based and cultivation approaches to search for chlamydiae in different freshwaters, including natural ponds, garden pots and fountains. From a total of 40 samples, we recovered 16 phylotypes, clustered into nine species-level taxa belonging to the Parachlamydiaceae, Simkaniaceae, Rhabdochlamydiaceae, cvE6 lineage and a novel lineage. Parachlamydiaceae (four species) were recovered by amoeba coculture, while the other chlamydiae were maintained in mixed eukaryotic cultures. This study confirms the widespread occurrence of novel chlamydiae in the environment and enlarges our knowledge of their biodiversity in freshwater habitats.