Mesopelagic fish belonging to the families Gonostomatidae, Phosichthyidae, Sternoptychidae, Stomiidae, Paralepididae, Myctophidae, Evermannellidae, Microstomatidae, and Nemichthyidae were sampled using an Isaacs Kidd Midwater Trawl (IKMT) in the Ligurian Sea, Northwestern Mediterranean, the core of the Cetacean Sanctuary established in 2001. In September–October 2002 and September 2003, 31 species of mesopelagic fish were captured in oblique hauls from 800 m depth to the surface. The mesopelagic fish fraction represented about 22% of the collected biomass, dominated by two main species of the genus Cyclothone, representing 93.8% of total abundance, followed by Argyropelecus hemigymnus and Lampanyctus crocodilus. The presence of Valenciennellus tripunctulatus was a new record for the study area. The family Myctophidae was the most represented in terms of number of species (n = 13). The abundance and biodiversity of mesopelagic fishes were influenced by the sampling period and oceanographic conditions, specifically temperature. While no diel vertical migrations could be observed, day and night samples revealed different compositions of juveniles and adults of Benthosema glaciale and Ceratoscopelus maderensis. The broad size range of specimens sampled confirmed that adult individuals of Chauliodus sloani and Lampanyctus crocodilus can inhabit and forage within the water column beyond the slope rather than exclusively near the bottom.