The North Temperate Lakes - Microbial Observatory seeks to study freshwater microbes over long time scales (10+ years). Observing microbial communities over multiple years using DNA sequencing allows in-depth assessment of diversity, variability, gene content, and seasonal/annual drivers of community composition. Combining information obtained from DNA sequencing with additional experiments, such as investigating the biochemical properties of specific compounds, gene expression, or nutrient concentrations, provides insight into the functions of microbial taxa. Our 16S rRNA gene amplicon datasets were collected from bog lakes in Vilas County, WI, and from Lake Mendota in Madison, WI. Ribosomal RNA gene amplicon sequencing of freshwater environmental DNA was performed on samples from Crystal Bog, North Sparkling Bog, West Sparkling Bog, Trout Bog, South Sparkling Bog, Hellâ€™s Kitchen, and Mary Lake. These microbial time series are valuable both for microbial ecologists seeking to understand the properties of microbial communities and for ecologists seeking to better understand how microbes contribute to ecosystem functioning in freshwater.