Observing multiple size classes of organisms, along with oceanographic properties and water mass origins, can improve our understanding of the drivers of aggregations, yet acquiring these measurements remains a fundamental challenge in biological oceanography. By deploying multiple biological sampling systems, from conventional bottle and net sampling to in situ imaging and acoustics, we describe the spatial patterns of different size classes of marine organisms (several microns to ∼10 cm) in relation to local and regional (m to km) physical oceanographic conditions on the Delaware continental shelf. The imaging and acoustic systems deployed included (in ascending order of target organism size) an imaging flow cytometer (CytoSense), a digital holographic imaging system (HOLOCAM), an In Situ Ichthyoplankton Imaging System (ISIIS, 2 cameras with different pixel resolutions), and multi-frequency acoustics (SIMRAD, 18 and 38 kHz). Spatial patterns generated by the different systems showed size-dependent aggregations and differing connections to horizontal and vertical salinity and temperature gradients that would not have been detected with traditional station-based sampling (∼9-km resolution). A direct comparison of the two ISIIS cameras showed composition and spatial patchiness changes that depended on the organism size, morphology, and camera pixel resolution. Large zooplankton near the surface, primarily composed of appendicularians and gelatinous organisms, tended to be more abundant offshore near the shelf break. This region was also associated with high phytoplankton biomass and higher overall organism abundances in the ISIIS, acoustics, and targeted net sampling. In contrast, the inshore region was dominated by hard-bodied zooplankton and had relatively low acoustic backscatter. The nets showed a community dominated by copepods, but they also showed high relative abundances of soft-bodied organisms in the offshore region where these organisms were quantified by the ISIIS. The HOLOCAM detected dense patches of ciliates that were too small to be captured in the nets or ISIIS imagery. This near-simultaneous deployment of different systems enables the description of the spatial patterns of different organism size classes, their spatial relation to potential prey and predators, and their association with specific oceanographic conditions. These datasets can also be used to evaluate the efficacy of sampling techniques, ultimately aiding in the design of efficient, hypothesis-driven sampling programs that incorporate these complementary technologies.