The copepod Calanus finmarchicus is one of the most abundant and ecologically important species of the zooplankton assemblage of the North Atlantic Ocean and occupies a pivotal position in the pelagic food web. This study used metabarcoding analysis (high throughput DNA sequencing of target gene regions) to examine the diversity of the copepod gut content, including both eukaryotic and prokaryotic components of the diet and microbiome. Zooplankton samples were collected during the 2013 EuroBASIN cruise of the R/V Go Sars, which crossed the North Atlantic to survey in the Norwegian, Icelandic, Irminger, and Labrador Seas. DNA was extracted from the dissected gut contents of adult female copepods and sequenced for eukaryotic 18S V4 and prokaryotic 16S V3-V4 rRNA hypervariable regions. The diverse assemblage revealed by metabarcoding analysis of copepod gut contents likely represents prey, microbiome, parasites, symbionts, and pathogens. Significant differences in prokaryotic and eukaryotic diversity of the gut contents of copepods collected from four regional seas of the North Atlantic Ocean reflect and contribute to basin-scale differences in the pelagic food web of these ecosystems.