Hurricane Florence brought unprecedented rainfall and flooding to Eastern North Carolina in 2018. Extensive flooding had the potential to mobilize microbial contaminants from a variety of sources. Our study evaluated microbial contaminants in surface waters at 40 sites across Eastern North Carolina 1 week after the hurricane made landfall (Phase 1) and one month later (Phase 2). High concentrations of Escherichia coli were detected in flowing channel and floodwater samples across both phases; however, channel samples during Phase 2 had higher concentrations of E. coli compared to Phase 1. Human- and swine-associated fecal markers were detected in 26% and 9% of samples, respectively, with no trends related to phase of sampling. Arcobacter butzleri was previously shown to be recovered from most (73%) samples, and detection of this pathogen was not associated with any source-associated fecal marker. Detection of Listeria spp. was associated with the swine-associated fecal marker. These results suggest that improved swine and human feces management should be explored to prevent microbial contamination in surface water, especially in regions where extreme rainfall may increase due to climate change. Sampling at higher frequency surrounding rainfall events would provide more detailed characterization of the risks posed by floodwater at different time scales and under different antecedent conditions.