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Microorganisms are ubiquitous in freshwater aquatic environments, but little is known about their abundance, diversity, and transport. We designed and deployed a remote-operated water-sampling system onboard an unmanned surface vehicle (USV, a remote-controlled boat) to collect and characterize microbes in a freshwater lake in Virginia, USA. The USV collected water samples simultaneously at 5 and 50 cm below the surface of the water at three separate locations over three days in October, 2016. These samples were plated on a non-selective medium (TSA) and on a medium selective for the genusPseudomonas(KBC) to estimate concentrations of culturable bacteria in the lake. Mean concentrations ranged from 134 to 407 CFU/mL for microbes cultured on TSA, and from 2 to 8 CFU/mL for microbes cultured on KBC. There was a significant difference in the concentration of microbes cultured on KBC across three sampling locations in the lake (P= 0.027), suggesting an uneven distribution ofPseudomonasacross the locations sampled. There was also a significant difference in concentrations of microbes cultured on TSA across the three sampling days (P= 0.038), demonstrating daily fluctuations in concentrations of culturable bacteria. There was no significant difference in concentrations of microbes cultured on TSA (P= 0.707) and KBC (P= 0.641) across the two depths sampled, suggesting microorganisms were well-mixed between 5 and 50 cm below the surface of the water. About 1 percent (7/720) of the colonies recovered across all four sampling missions were ice nucleation active (ice+) at temperatures warmer than −10 °C. Our work extends traditional manned observations of aquatic environments to unmanned systems, and highlights the potential for USVs to understand the distribution and diversity of microbes within and above freshwater aquatic environments.