Vibrio cholerae is a natural inhabitant of aquatic ecosystems worldwide, typically residing in coastal or brackish water. While more than 200 serogroups have been identified, only serogroups O1 and O139 have been associated with epidemic cholera. However, infections other than cholera can be caused by nonepidemic, non-O1/non-O139 V. cholerae strains, including gastroenteritis and extraintestinal infections. While V. cholerae can also survive in freshwater, that is typically only observed in regions of the world where cholera is endemic. We recently isolated V. cholerae from several locations in lakes and rivers in northwest Ohio. These isolates were all found to be non-O1/non-O139 V. cholerae strains, that would not cause cholera. However, these isolates contained a variety of virulence genes, including ctxA, rtxA, rtxC, hlyA, and ompU. Therefore, it is possible that some of these isolates have the potential to cause gastroenteritis or other infections in humans. We also investigated the relative motility of the isolates and their ability to form biofilms as this is important for V. cholerae survival in the environment. We identified one isolate that forms very robust biofilms, up to 4x that of our laboratory strains. Finally, we investigated the susceptibility of these isolates to a panel of antibiotics. We found that many of the isolates showed decreased susceptibility to some of the antibiotics tested, which could be of concern. While we do not know if these isolates are pathogenic to humans, increased surveillance to better understand the public health risk to the local community should be considered.