The dataset is composed of six tables containing environmental DNA (eDNA) and electrofishing data from American eel (Anguilla rostrata) surveys conducted on parts of the Mohawk River watershed and tributaries to the Hudson River in New York. The dataset includes (a) eDNA data from 36 sites in the Mohawk River watershed and adjacent areas including parts of the Eastern Erie Canal, Mohawk River, and tributaries that were sampled in spring 2021 and again in summer 2021,(b) paired eDNA/electrofishing data from 15 sites on tributaries to the Hudson River that were sampled between 2020 and 2021, and (c) model output of predicted American eel density from all sites in the Mohawk River watershed and adjacent areas where eDNA was detected. The table "MohawkHudsonEel_MohawkScreening_eDNAData" contains eDNA data from the 36 sites in the Mohawk River watershed, the table "MohawkHudsonEel_MohawkScreening_SiteData" contains supporting information on the 36 sites in the Mohawk River watershed, the table "MohawkHudsonEel_HudsonTribCalibration_LWData" contains the length and weight for all American eel captured during electrofishing surveys from the 15 sites on tributaries to the Hudson River, the table "MohawkHudsonEel_HudsonTribCalibration_eDNAData" contains eDNA data from the 15 sites on tributaries to the Hudson River, the table "MohawkHudsonEel_HudsonTribCalibration_SiteData" contains supporting information on the 15 sites on tributaries to the Hudson River including the dimensions of the reaches where electrofishing was conducted, and the table "MohawkHudsonEel_MohawkScreening_DensityEstimate" contains predicted density of American eel estimated from eDNA concentration using linear regression from all sites in the Mohawk River watershed and adjacent areas where eDNA was detected.