Gelatinous zooplankton play a crucial role in marine planktonic food webs. However, primarily due to methodological challenges, the in situ diet of zooplankton remains poorly investigated and little is known about their trophic interactions including feeding behavior, prey selection, and in situ feeding rates. This is particularly true for gelatinous zooplankton including the marine pelagic tunicate, Dolioletta gegenbauri. In this study, we applied an 18S rRNA amplicon metabarcoding approach to identify the diet of captive-fed and wild-caught D. gegenbauri on the mid-continental shelf of the South Atlantic Bight (SAB), USA. Sequencing-based approaches were complimented with targeted quantitative real time PCR (Polymerase Chain Reaction) analyses. Captive-fed D. gegenbauri gut content was dominated by pico-, nano- and microplankton including picodinoflagellates (picozoa) and diatoms. These results suggested that diatoms were concentrated by D. gegenbauri relative to their concentration in the water column. Analysis of wild-caught doliolids by quantitative real time PCR utilizing a group-specific diatom primer set confirmed that diatoms were concentrated by D. gegenbauri, particularly by the gonozooid life stage associated with actively developing blooms. Sequences derived from larger metazoans were frequently observed in wild-caught animals but not in captive-fed animals suggesting experimental bias’ associated with captive feeding. These studies revealed that the diet of D. gegenbauri is considerably more diverse than previously described, that parasites are common in wild populations, and that prey quality, quantity and parasites are likely all important factors in regulating doliolid population dynamics in continental shelf environments.