Aquatic surface microlayers are unique microbial ecosystems found at the air–water interface of all open water bodies and are often referred to as the neuston. Unambiguous interpretation of the microbiology of aquatic surface microlayers relies on robust sampling, for which several methods are available. All have particular advantages and disadvantages that make them more or less suited to this task. A key feature of surface microlayers is their role in regulating air–water gas exchange, which affords them a central role in global biogeochemistry that is only now being fully appreciated. The microbial populations in surface microlayers can impact air–water gas exchange through specific biogeochemical processes mediated by particular microbial groups such as methanotrophs or through more general metabolic activity such as the balance of primary production vs. heterotrophy. There have been relatively few studies of surface microlayers that have utilized molecular ecology techniques. The emerging consensus view is that aquatic surface microlayers are aggregate-enriched biofilm environments containing complex microbial communities that are ecologically distinct from those present in the subsurface water immediately below. Future research should focus on unravelling the complex interactions between microbial diversity and the ecosystem function of surface microlayers in order to better understand the important but complex role of microorganisms in Earth system processes.