Despite its ecological importance, micronekton remains one of the least investigated components of the open-ocean ecosystems. Our main goal was to characterize micronektonic organisms using bi-frequency acoustic data (38 and 120 kHz) by calibrating an algorithm tool that discriminates groups of scatterers in the top 300 m of the productive oceanic zone east of Kerguelen Islands (Indian sector of the Southern Ocean). The bi-frequency algorithm was calibrated from acoustic properties of mono-specific biological samples collected with trawls, thus allowing to discriminate three acoustic groups of micronekton: (i) “gas-bearing” (ΔS_v,120-38 < -1 dB), (ii) “fluid-like” (ΔS_v,120-38 > 2 dB), and (iii) “undetermined” scatterers (-1 < ΔS_v,120-38 < 2 dB). The three groups likely correspond biologically to gas-filled swimbladder fish (myctophids), crustaceans (euphausiids and hyperiid amphipods), and other marine organisms potentially present in these waters and containing either lipid-filled or no inclusion (e.g. other myctophids), respectively. The Nautical Area Scattering Coefficient (NASC) was used (echo-integration cells of 10m long and 1m deep) between 30 and 300m depth as a proxy of relative biomass of acoustic targets. The distribution of NASC values showed a complex pattern according to: (i) the three acoustically-defined groups, (ii) the type of structures (patch vs. layers) and (iii) the timing of the day (day/night cycle). NASC values were higher at night than during the day. A large proportion of scatterers occurred in layers while patches, that mainly encompass gas-bearing organisms, are especially observed during daytime. This method provided an essential descriptive baseline of the spatial distribution of micronekton and a relevant approach to (i) link micronektonic group to physical parameters to define their habitats, (ii) investigate trophic interactions by combining active acoustic and top predator satellite tracking, and (iii) study the functioning of the pelagic ecosystems at various spatio-temporal scales.