We sampled plankton from 55 freshwater peri-alpine lakes in the northern Alps region in Europe (Austria, Germany, Switzerland) in 2011 and 2012. Five environmental variables were recorded for each lake: water temperature (ºC), chlorophyll a concentration (μg/l), electrical conductivity (µS/cm), lake area (hectares), and altitude (m). These variables were used as proxies of the lakes’ main environmental characteristics and the level of anthropogenic influence. Sampling was carried out by a boat, with smaller lakes being sampled above their deepest points, and larger lakes at depths >15m, with the exception of Hubertussee, where water was collected from the outlet of the lake. Water samples were collected by a tube sampler from the upper mixed layer (i.e., epilimnion), following inspection of temperature profiles which were measured prior to collecting the water samples. Water samples were filtered onto 0.2 micrometer pore size membrane filters. DNA was extracted from the filters using the MoBio PowerSoil DNA isolation kit and fragments of the 16S- and 18S rRNA genes of bacteria and eukaryotes were amplified and sequenced using Illumina MiSeq technology.