Diel vertical migration (DVM) facilitates biogeochemical exchanges between shallow waters and the deep ocean. An effective way of monitoring the migrant biota is by acoustic observations although the interpretation of the scattering layers poses challenges. Here we combine results from acoustic observations at 18 and 38 kHz with limited net sampling in order to unveil the origin of acoustic phenomena around the Canary Islands, subtropical northeast Atlantic Ocean. Trawling data revealed a high diversity of fishes, decapods and cephalopods (152 species), although few dominant species likely were responsible for most of the sound scattering in the region. We identified four different acoustic scattering layers in the mesopelagic realm: (1) at 400–500 m depth, a swimbladder resonance phenomenon at 18 kHz produced by gas-bearing migrant fish such as Vinciguerria spp. and Lobianchia dofleini, (2) at 500–600 m depth, a dense 38 kHz layer resulting primarily from the gas-bearing and non-migrant fish Cyclothone braueri, and to a lesser extent, from fluid-like migrant fauna also inhabiting these depths, (3) between 600 and 800 m depth, a weak signal at both 18 and 38 kHz ascribed either to migrant fish or decapods, and (4) below 800 m depth, a weak non-migrant layer at 18 kHz which was not sampled. All the dielly migrating layers reached the epipelagic zone at night, with the shorter-range migrations moving at 4.6 ± 2.6 cm s − 1 and the long-range ones at 11.5 ± 3.8 cm s − 1. This work reduces uncertainties interpreting standard frequencies in mesopelagic studies, while enhances the potential of acoustics for future research and monitoring of the deep pelagic fauna in the Canary Islands.