Sharp boundaries in the physical environment are usually associated with abrupt shifts in organism abundance, activity, and diversity. Aquatic surface microlayers (SML) form a steep gradient between two contrasted environments, the atmosphere and surface waters, where they regulate the gas exchange between both environments. They usually harbor an abundant and active microbial life: the neuston. Few ecosystems are subjected to such a high UVR regime as high altitude lakes during summer. Here, we measured bulk estimates of heterotrophic activity, community structure and single-cell physiological properties by flow cytometry in 19 high-altitude remote Pyrenean lakes and compared the biological processes in the SML with those in the underlying surface waters. Phototrophic picoplankton (PPP) populations, were generally present in high abundances and in those lakes containing PPP populations with phycoerythrin (PE), total PPP abundance was higher at the SML. Heterotrophic nanoflagellates (HNF) were also more abundant in the SML. Bacteria in the SML had lower leucine incorporation rates, lower percentages of "live" cells, and higher numbers of highly-respiring cells, likely resulting in a lower growth efficiency. No simple and direct linear relationships could be found between microbial abundances or activities and environmental variables, but factor analysis revealed that, despite their physical proximity, microbial life in SML and underlying waters was governed by different and independent processes. Overall, we demonstrate that piconeuston in high altitude lakes has specific features different from those of the picoplankton, and that they are highly affected by potential stressful environmental factors, such as high UVR radiation.