Oxygen-dependent, neutrophilic iron-oxidizing bacteria (FeOB) are important drivers of iron transformations in marine and freshwater environments. Despite remarkable similarities in physiology and morphotype, known freshwater and marine FeOB are clustered in different classes of Proteobacteria; freshwater FeOB in the Betaproteobacteria and marine FeOB in the Zetaproteobacteria. To determine effects of salinity on these microbes, we examined the mineral biosignatures and molecular ecology of bacteria in FeOB mats collected along an estuarine salinity gradient. Light microscopy and scanning electron microscopy analyses showed the presence of iron oxide stalk and sheath structures in both freshwater and saline iron mats. Results of tagged pyrosequencing, quantitative PCR and fluorescent in situ hybridization, all based on the small subunit rRNA gene, confirmed Zetaproteobacteria were not present in freshwater mats, but were in saline mats at salinities down to 5‰. Among the Betaproteobacteria, Leptothrix spp. were only found in the freshwater mat. Gallionella spp. were limited to freshwater and low salinity mats (< 5‰). Sideroxydans sp. were salt tolerant; however, their relative abundance decreased with increasing salinity. These results suggest salinity is important in shaping the population biology of iron mat communities, and some coexistence between marine and freshwater populations occurs in brackish waters.