Mesopelagic fish constitute the most abundant vertebrate group in the marine environment. The current work reports on results of three seasonal acoustic cruises carried out in the Gulf of Corinth, a relatively small, deep, isolated basin located in the Central Mediterranean (Greece) that presents some unique geomorphological and ecological features. The aim of this study was to describe seasonal echo-types and the vertical distribution of the Deep Scattering Layers (DSLs) as well as to relate them with specific species or species groups. Mesopelagic fish dominated the pelagic ecosystem as confirmed by biological sampling with different gears during daytime and nighttime. In total, at least 15 species were caught, belonging to the families Myctophidae, Paralepididae, Sternoptychidae and Stomiidae, while the—elsewhere very abundant—families Gonostomatidae and Phosichthyidae were completely absent. Common echo-types included: (a) shoals and schools formed by the silvery lightfish Maurolicus muelleri, usually located along the shelf break (80–225 m), (b) a non-migrant thin DSL found at 150–280 m throughout the deep parts of the Gulf, dominated by juvenile half-naked hatchetfish Argyropelecus hemigymnus, and (c) one thick, partially migratory DSL at 250–600 m, mainly consisting of myctophids. The echo backscatter characteristics and species composition of the DSLs as well as the length distribution of the populations were found to differ seasonally. Species-specific and size related patterns in the vertical distribution of fish were detected both during daytime and nighttime. Overall, the Gulf of Corinth seems to sustain high densities of mesopelagic fish that constitute the basic food resource for the abundant dolphin populations that inhabit the area.