An uncrewed surface vessel, USV Surveyor, and an automated eDNA sample collection device, the Environmental Sample Processor, were used to obtain eDNA samples autonomously over a 4,200 km, 29 day voyage in the eastern North Pacific Ocean. We used DNA metabarcoding (Illumina MiSeq) of cytochrome c oxidase subunit 1 (COI) to investigate the eukaryotic diversity across the transect. Also included in the data set are control samples including positive and negative controls collected by the autonomous sampler, positive controls samples collected by standard laboratory methods, extraction blanks, and no template controls from PCR amplification.