Surface microlayers (SMLs) play a key role in regulating air-water gas exchange. Few studies have investigated bacterial diversity in this particular zone in freshwater ecosystems compared to that of the epilimnetic water layer, using high-throughput sequencing technologies. The present study examined bacterial community structures in the SML and the epilimnion layers of an oligotrophic high mountain lake, Lake Pavin, France. These 2 habitats harboured distinct bacterial communities, with higher temporal variation observed in the structure of the bacterial community in the SML than in the epilimnion. This arrangement might result from aerosol deposition and/or different levels of solar radiation impacting the lake surface. The presence of typical freshwater bacterial assemblages (i.e. Alphaproteobacteria LD12 lineage, and Actinobacteria AcI clade) in the epilimnion was also examined. In contrast, the SML was clearly enriched in Alpha proteo-bacteria (i.e. Rhodospirillales and Sphingomonadales), Gammaproteobacteria (i.e. Pseudo-monadales), and Firmicutes (i.e. Clostridiales). Notably, analyses conducted on indicator species revealed that Propionibacterium and Acinetobacter were characteristic in the SML, whereas operational taxonomic units in the epilimnion showed greater diversity. SMLs present a specific bacterial community composition that might originate from the surrounding environment, raising questions about the metabolic adaptations of these organisms in this particular environment.