BackgroundLegionellaare predominantly recognized as aerobic pathogens in man-made water systems. However, their potential persistence in natural freshwater environments, particularly under oxygen limited conditions, remains poorly explored. In this study we investigated the spatio-temporal dynamics ofLegionellaoccurrence in a seasonally stratified sub-tropical freshwater lake, with a focus on anaerobic conditions of the anoxic hypolimnion.ResultsOur study reveals significant seasonal variations inLegionellaabsolute abundance, with the highest concentrations occurring during and immediately following lake mixing events. Unexpectedly, high levels ofLegionellawere measured in the anaerobic hypolimnion layer of the lake. Utilizing genus specific amplicon-based sequencing, we found significant shifts inLegionellacommunity composition, that are related to the sampling month. Several environmental factors were associated with the observed changes, including temperature, DO, chlorophyll and dinoflagellate biomass. Moreover, we identifiedLegionellagenotypes unique to samples presenting hypoxic conditions - that were not closely related to knownLegionellaspecies. In addition, we noted genotypes present in anoxic samples, that were absent from the oxic layers of the corresponding sampling dates. These results were accompanied by changes in the interaction patterns betweenLegionellaand their potential hosts, in oxic and anoxic conditions.ConclusionsThis study challenges the conventional view ofLegionellaas a strictly aerobic pathogen by demonstrating its persistence in anoxic freshwater environments. Our findings suggest that certainLegionellaspecies may have adapted to low- oxygen conditions, potentially through alternate metabolic pathways or by residing within protozoan hosts. The identification of uniqueLegionellagenotypes in the hypolimnion, along with shifts in occurrence, diversity, and host interactions, underscores the complexity ofLegionellaecology. These results highlight the need for further research onLegionellain natural freshwater systems, which serve as reservoirs for the bacteria and potential sources for human infection. Further investigation into the mechanisms underlyingLegionellapersistence in anaerobic conditions and its interactions with environmental hosts is essential for a better understanding of the evolutionary forces shaping this family of human pathogens.