Interest in mesopelagic fish layers is on the rise due to the potential exploitability of their macrofauna; hence, profiling their fish community is crucial to enable the sustainability of future fishing practices. In this context, a dedicated survey was carried out in September 2022 along the Irish shelf break, where fishing (catch) and eDNA metabarcoding analysis using a portable high-throughput sequencer were performed to investigate the fish community of the upper mesopelagic layer. Catch data showed that the targeted layers consisted mainly of the young-of-the-year cohort of Mueller’s pearlside (Maurolicus muelleri), a mesopelagic fish, with little bycatch. eDNA data reflected the high prevalence of M. muelleri’s eDNA (56%–97% of assigned reads), identified species undetected by fishing, and showed that the least represented species differed in water samples collected before or after fishing activities. While this reflects current limitations of each technique, it also shows that a multidisciplinary approach may provide an increased level of resolution for M. muelleri layer’s ancillary fish community. Findings from the present study provided important insights to further refine sample acquisition and rapid processing of eDNA metabarcoding data, which beholds great potential to corroborate fishing methods when ground truthing acoustic approaches in mesopelagic fish layers assessments.