Many estuarine systems are chronically contaminated with fecal material, as assessed by fecal indicator bacteria (FIB). These FIB counts cannot resolve the source of the contamination, particularly in coastal systems that receive significant input of waste from multiple sources. Western Galveston Bay receives input from tributaries that are coupled to mixed-use watersheds in the Houston-Galveston area. These watersheds include suburban communities with high numbers of on-site sewage facilities (OSSF), which are a suspected source of fecal contamination. Culture-independent microbial source tracking (MST) methods, including metagenomic analysis and amplification of source-specific markers, can identify the source of fecal contamination in aquatic systems but these molecular methods are rarely applied to water quality monitoring. In this study, we used digital PCR and high-throughput 16S rRNA gene amplicon sequencing to identify human-specific markers across tidal and non-tidal waters of the Houston-Galveston area. Enterococcus spp. and crAssphage, a human-specific bacteriophage, concentrations were quantified via dPCR. DADA2 was used to process reads generated by high-throughput sequencing, and SourceTracker2 was used to estimate the proportion of fecal sources. Enterococcus spp. counts were elevated in tidal sites and crAssphage were elevated in non-tidal sites. Multivariate analysis suggested that crAssphage abundance and dissolved inorganic nutrient concentrations were linked to bacterial community structure in non-tidal sites. Non-tidal sites also showed relatively high human-associated taxa and metabolic functions and SourceTracker2 suggested a low but detectable contribution from human waste in these water bodies. This suggests that human waste, perhaps from failing OSSF, is likely partially responsible for elevated FIB counts in the non-tidal areas of tributaries to Western Galveston Bay.