Environmental DNA (eDNA) analysis has become a widespread conservation tool. One promising direction is to evaluate the impacts of anthropogenic disturbances on wildlife populations. In 2016, a ruptured pipeline caused 207,000 L of gasoline to spill into a tributary of the West Branch Susquehanna River in central Pennsylvania. This tributary is known to harbor one of Pennsylvania's most prolific populations of Cryptobranchus alleganiensis alleganiensis (Eastern Hellbender), a giant salamander of special conservation concern. To evaluate the impact of the gasoline spill on Hellbender populations, we conducted spatial eDNA analysis on 9 water samples collected along a 20-km stretch of the tributary in 2018. Spatial analysis identified a notably low eDNA concentration at the site immediately downstream of the spill site, likely a result of habitat destruction caused by the flood, gasoline spill, and subsequent construction of a new bridge and stream bank. We found relatively high eDNA concentrations at several sampled sites, suggesting the gasoline spill did not completely extirpate Hellbender populations from the tributary. Finally, we make recommendations on sampling regimes, including proactive sampling, to encourage eDNA-based assessments of the impacts of future spill events on wildlife populations.