The Northern Humboldt Current system (NHCS) produces more fish per unit area than any other region in the world. Although the system produces enough macrozooplankton to sustain its high production of forage fish, the paucity of information on macrozooplankton hampers research into the system. In this study, we estimated the biomass of the epipelagic crustacean macrozooplankton from the NHCS during both austral summer and spring 2005. To do this, we developed a bi-frequency acoustic method and extracted high-resolution information on the biomass and the patterns of distribution of crustacean macrozooplankton, fish and other marine compartments. We found that, although macrozooplankton comprises a number of distinct organisms, the euphausiids were the zooplankton group that better fitted the patterns from independent net sampling zooplankton data. Also, the similarities between the nocturnal patterns of size and biomass macrozooplankton distribution from this study and the known patterns of euphausiids, in particular Euphausia mucronata, suggest that euphausiids were the main constituent of the estimated nocturnal acoustic macrozooplankton biomass even if other organisms such as large copepods may have contributed considerably to the macrozooplankton biomass. The total macrozooplankton biomass was estimated to about 105gm-2, i.e., two to five times more than previous estimates. This direct biomass estimation of macrozooplankton is in agreement with the new findings in trophic ecology indicating that forage fish consume mainly macrozooplankton. This high biomass also supports the current hypotheses explaining the NHCS high fish production. Using the method, we are able to revisit present-day and historical acoustic databases and extract high-resolution data on macrozooplankton, a key ecological compartment of the ecosystem. Since zooplankton is the link between the physically driven primary producers and the biologically driven tertiary consumers, this information is essential to achieve a mechanistic understanding of the system, from physics to top predators. © 2011 Elsevier Ltd.