Most coastal freshwater ecosystems in the United States have semi-tidal movements during the day. Routine monitoring of these environments is conducted once during the day when tides can be at either ebb or flood conditions, causing a variability in bacterial concentrations and misinterpretation of the illness risk associated with human activities. The occurrence and levels of enterococci (enterococci 23S rDNA [Ent23S]) and human- (HF183) and avian- (GFD) associated microbial source tracking (MST) markers were investigated using quantitative polymerase chain reaction (qPCR) along with detection of culturable enterococci and environmental parameters. Samples were collected during flood and ebb tide conditions (May–September) from a tidal river used for recreational activities. Culturable enterococci [t(420) = 2.093, p = 0.040] and Ent23S [t(420) = 2.243, p = 0.028] controlled for tide type were significantly different; higher enterococci concentrations were detected during the flood tide. Among all samples, 6% were positive for HF183, and GFD was positively correlated with Ent23S (r = 0.92, p = 0.029) and conductivity (r = 0.93, p = 0.023) during flood tide. Unlike the general assumption that ebb tide flow in a river would likely carry runoff from the land, the microbial contaminants in this case were transported from upstream via ocean water to the river during the flood tide. These results suggest that hydrology and land use patterns must be considered in sampling design when conducting future microbial water quality monitoring programs to better characterize recreational water safety in tidal rivers.