<jats:p> The South Pacific Gyre, due to its vast size and remoteness, is one of the least-studied oceanic regions on earth. However, both remote sensing and <jats:italic>in situ</jats:italic> measurements indicated that the activity of its microbial community contributes significantly to global biogeochemical cycles. Presented here is an unparalleled investigation of the microbial community of the SPG from 20- to 5,000-m depths covering a geographic distance of ∼7,000 km. This insight was achieved through the development of a novel onboard analysis pipeline, which combines next-generation sequencing with fluorescence <jats:italic>in situ</jats:italic> hybridization and automated cell enumeration. The pipeline is well comparable to onshore systems based on the Illumina platforms and yields microbial community data in less than 35 h after sampling. Going forward, the ability to gain on-site knowledge of a remote microbial community will permit hypothesis-driven research, through the generation of novel scientific questions and subsequent additional targeted sampling efforts. </jats:p>