In this data paper, we describe environmental DNA (eDNA) cytochrome c oxidase (COI) amplicon sequence data from New York City’s Bronx River Estuary. As urban systems continue to expand, describing and monitoring their biodiversity is increasingly important for sustainability. Once polluted and overexploited, New York City’s Bronx River Estuary is undergoing revitalization and restoration. To investigate and characterize the area’s diversity, we collected and sequenced river sediment and surface water samples from Hunts Point Riverside and Soundview Parks (n total = 48; n sediment = 25; n water = 23). COI analysis using universal primers mlCOIintF and jgHCO2198 detected 27,328 Amplicon Sequence Variants (ASVs) from 7,653,541 sequences, and rarefaction curves reached asymptotes indicating sufficient sampling depth. Of these, eukaryotes represented 9,841ASVs from 3,562,254 sequences. At the study sites over the sampling period, community composition varied by substrate (river sediment versus surface water) and with water temperature, but not pH. The three most common phyla were Bacillariophyta (diatoms), Annelida (segmented worms), and Ochrophyta (e.g. brown and golden algae). Of the eukaryotic ASVs, we identified 614 (6.2%) to species level, including several dinoflagellates linked to Harmful Algal Blooms such as Heterocapsa spp., as well as the invasive amphipod Grandidierella japonica . The analysis detected common bivalves including blue ( Mytilus edulis ) and ribbed ( Geukensia demissa ) mussels, as well as soft-shell clams ( Mya arenaria ), in addition to Eastern oysters ( Crassostrea virginica ) that are being reintroduced to the area. Fish species undergoing restoration such as river herring ( Alosa pseudoharengus , A. aestivalis ) failed to be identified, although relatively common fish including Atlantic silversides ( Menidia menidia ), menhaden ( Brevoortia tyrannus ), striped bass ( Morone saxatilis ), and mummichogs ( Fundulus heteroclitus ) were found. The data highlight the utility of eDNA metabarcoding for analyzing urban estuarine biodiversity and provide a baseline for future work in the area.