A comprehensive survey to document the presence of free-living amoebae was conducted along 58 km of James River, near Richmond, Virginia, USA. Sites included tidal and non-tidal freshwater areas, near 40 combined sewer outflows, three municipal wastewater treatment plant release sites, and thermal discharge from a coal-fired power plant. Amoebae were present on all collection dates, spring through autumn, and at all sites (n=330). Five genera, Naegleria, Vannella, Acanthamoeba, Vahlkampfia, and Hartmannella were present in both the water column and sediment. The most common isolates from the water column were Naegleria and Vannella. Water conditions conducive to the presence of large quantities of fecal coliform bacteria were correlated with the prevalence of free-living amoebae. Some of the amoebae in this complex ecosystem can act as opportunistic pathogens, may play a role in diseases of aquatic organisms in this heavily urbanized river, and may present a risk to human health.