Studies of the species composition, relative abundance and biomass of deep-sea assemblages are needed to create a knowledge base for the assessment of the resource potential and to advise ecosystem-based conservation and fisheries management strategies. This study attempts to create an initial knowledge base of the system by assessing the deep-sea fish assemblages of Colombian Caribbean Sea across a depth range of 200–550 m. A total of 102 species (13 Chondrichthyes and 89 Teleosteans) and 58 families (9 Chondrichthyes and 49 Teleosteans) of deep-sea fish were sampled. This study reveals the existence of three deep-sea fish assemblages and demonstrates that depth plays an important role in ichthyofaunal group differentiation. Most species of the shallower assemblage exhibited a narrow depth range, while species of middle depth assemblage showed the widest depth range. From the point of view of conservation and fisheries management strategies, we need to know the status of deep-sea fish assemblages more fully before initiating a new deep-sea fishery in the Colombian Caribbean Sea. Conservation and protection of the habitats is therefore fundamental to develop a rational and sustainable management of the deep-sea fishery resources of the Colombian Caribbean Sea.