Surface water contaminants in Kentucky during and after 2011 flooding were characterized. Surface water samples were collected during flood stage (May 2–4, 2011; n = 15) and after (July 25–26, 2011; n = 8) from four different cities along the Ohio River and were analyzed for the presence of microbial indicators, pathogens, metals, and chemical contaminants. Contaminant concentrations during and after flooding were compared using linear and logistic regression. Surface water samples collected during flooding had higher levels of E. coli, enterococci, Salmonella, Campylobacter, E. coli O157:H7, adenovirus, arsenic, copper, iron, lead, and zinc compared to surface water samples collected 3-months post-flood (P < 0.05). These results suggest that flooding increases microbial and chemical loads in surface water. These findings reinforce commonly recommended guidelines to limit exposure to flood water and to appropriately sanitize contaminated surfaces and drinking wells after contamination by flood water.