Zooplankton analysis represents a bottleneck in marine ecology studies due to the difficulty to obtain zooplankton data. The last decades have seen the intense development of zooplankton imaging systems, to increase the zooplankton data spatio-temporal resolution as well as enabling the combination of size, taxonomy, and functional traits in aquatic ecology studies. Here, we propose a benchmark between the ZooScan, a commercially available, laboratory-based scanner, which analyses zooplankton preserved samples, and the ZooCAM, an in-flow imaging system designed for on-board live zooplankton imaging. Sixty-one zooplankton samples collected over the Bay of Biscay in environments ranging from estuarine to offshore blue waters were imaged with both instruments. Zooplankton Normalized Biovolume-Size Spectra slopes, mean sizes, abundances, and zooplankton community biogeographical patterns were computed for each instrument and compared at the taxonomic group, the sampling stations and the Bay of Biscay scales. Both instruments produced similar zooplankton variables by stations and by taxa and described similar zooplankton community compositions and biogeographical patterns, on the large mesozooplankton size range, i.e., [0.3–3.39] mm ESD. We conclude that the ZooCAM and the ZooScan data can be combined to generate long term or spatially resolved zooplankton time series. Our study shows that benchmarking imaging instruments or techniques (1) offers a robust assessment of interoperability between instruments, mitigating possible instrumental biases, and (2) may be of great interest in the case of instrumental obsolescence or breakdown, to choose the most conservative replacement solution in a long term time series framework