Sound scattering layers (SSLs) are observed over a broad range of spatio-temporal scales and geographical areas. SSLs represent a large biomass, likely involved in the biological carbon pump and the structure of marine trophic webs. Yet, the taxonomic composition remains largely unknown for many SSLs. To investigate the challenges of SSL sampling, we performed a survey in a small study area in the Northern Bay of Biscay (France) by combining broadband and narrowband acoustics, net sampling, imagery and video recordings. In order to identify organisms contributing to the observed SSLs, we compared measured frequency spectra to forward predicted spectra derived from biological data. Furthermore, to assess the confidence in SSL characterization, we evaluated uncertainties in modeling, acoustical and biological samplings. Here, we demonstrate for the first time that SSL backscattering intensity in the Bay of Biscay can be dominated in springtime by resonant gas bearing organisms below 100 kHz, namely siphonophores and juvenile fishes and by pteropods at higher frequencies. Thus, we demonstrate the importance of broadband acoustics combined to nets, imagery and video to characterize resonant backscatterers and mixed mesozooplankton assemblages.