High-throughput sequencing has provided unprecedented insights into microbial biodiversity in marine and other ecosystems. However, most sequencing-based studies report only relative (compositional) rather than absolute abundance, limiting their application in ecological modeling and biogeochemical analyses. Here, we present a metagenomic protocol incorporating genomic internal standards to quantify the absolute abundances of prokaryotes and eukaryotic phytoplankton, which together form the base of the marine food web, in unfractionated seawater. We applied this method to surface waters collected across 50°N to 40°S during the 29 Atlantic Meridional Transect. Using the single-copy gene, we estimated an average bacterial abundance of 1.0 × 10 haploid genome equivalents per liter. Leveraging a recent report that the gene is typically single-copy in phytoplankton, we also quantified eukaryotic phytoplankton. Metagenomic estimates closely aligned with flow cytometry data for cyanobacteria (slope = 1.03, Pearson's  = 0.89) and eukaryotic phytoplankton (slope = 0.72, Pearson's  = 0.84). Compared to flow cytometry, taxonomic resolution for nano- and picoeukaryotes was greatly improved. Estimates for diatoms, dinoflagellates, and were considerably higher than microscopy counts, likely reflecting microscopy undercounts and potential ploidy variation. These findings highlight the value of absolute quantification by metagenomics and offer a robust framework for quantitative assessments in microbial oceanography.