Seawater was collected on board the NOAA ship Ronald H. Brown as part of the fourth Gulf of Mexico Ecosystems and Carbon Cycle (GOMECC-4) cruise from September 13 to October 21, 2021. Sampling for GOMECC-4 occurred along 16 coastal-offshore transects across the entire Gulf of Mexico and an additional line at 27N latitude in the Atlantic Ocean. We also collected eDNA samples near Padre Island National Seashore (U.S. National Parks Service), a barrier island located off the coast of south Texas. Vertical CTD sampling was employed at each site to measure discrete chemical, physical, and biological properties. Water sampling for DNA filtration was conducted at 54 out of 141 total sites and three depths per site (surface, deep chlorophyll maximum, and near bottom) to capture horizontal and vertical gradients of bacterial, protistan, and metazoan diversity across the Gulf.