Enterococci are fecal indicator bacteria (FIB) that are used worldwide for water quality assessment. However, evidence of high densities and extended survival of enterococci in sediments and submerged aquatic vegetation (SAV) has caused uncertainty about their reliability in predicting human health risks from recreational activities in environmental waters. To address the concern that sediments and SAV may harbor large reservoirs of enterococci that can affect water column concentrations, aquatic mesocosms and environmental sampling were employed to investigate patterns of enterococci densities and population structure across the Tampa Bay watershed. In mesocosm experiments and environmental samples, SAV harbored higher densities of enterococci, per mass of substrate, than sediments, and sediments harbored higher densities than water. Population structure assessed by BOX-PCR genotyping was relatively unique in each sample, although slight similarities among samples suggested grouping primarily by location rather than substrate or season. Strain diversity was highly variable, and many samples had low diversity, including nearly monoclonal structure throughout the mesocosm experiments and in several of the environmental samples. Several strains were highly abundant and cosmopolitan (found across sites, seasons, and substrates), and may represent highly naturalized and reproducing indicator bacteria populations that are not directly related to pollution events. When the enterococci densities were viewed from the perspective of the entire aquatic system, SAV-associated enterococci did not comprise a major proportion of the total population, due to the typically large differences in volume of each substrate (SAV vs. sediments vs. water). Instead, the largest proportions of enterococci were typically found in the water or the sediments, depending on the relative volume of substrate or the enterococci density associated with each substrate. Modeling results illustrate that the relative importance of each substrate in terms of FIB populations can shift dramatically over time and space due to changes such as vegetation cover, tidal cycles, and bacteria densities. Furthermore, at several sites within the watershed, estimates of sediment and bacteria resuspension from sediments were very low, suggesting that this process rarely, if ever, significantly affects water column concentrations of enterococci in the watershed.