These metabarcoding data were collected from bulk marine zooplankton samples at station ALOHA in the North Pacific Subtropical Gyre. Plankton from 9 depth strata were quantitatively split, and size-fractionated into 0.2-0.5m, 0.5-1.0mm, and 1.0-2.0mm size fractions. DNA was extracted in bulk, followed by PCR and amplicon sequencing of a ~ 356-bp fragment of the V1-V2 region of 18S rRNA (Fonseca et al. 2010 primers). The objectives of the study were to (i) assess the potential magnitude of undescribed metazoan diversity in poorly-characterized midwater habitats, (ii) detect distinct faunal assemblages and identify important faunal boundaries across depth in the twilight zone, (iii) identify suites of species that maximally distinguish these faunal assemblages, and (iv) test the expectation of a mesopelagic maximum in species richness for the full zooplankton assemblage using a metabarcoding approach.