Luminescent bacteria are a fascinating component of marine microbial communities, often related to the light emissions in deep sea marine organisms. They are mainly affiliated with specific phylogenetic groups, such as Photobacterium, Vibrio, and Photorhabdus, and are sometimes involved in symbiotic relationships. However, the luminescence of some marine organisms remains a poorly understood process, and it is not always certain whether their luminescence is attributable to associated luminescent bacteria. In this study, for the first time, luminescent bacteria were isolated from two deep sea organisms, namely, the cephalopod Neorossia caroli and the teleost Chlorophthalmus agassizi. The isolation was carried out on glycerol-supplemented medium, and the search for the luxAB gene was performed on all isolates as a complementary tool to the culture-dependent techniques to detect bioluminescence by molecular approach. The optimum of salinity, temperature, and pH was evaluated by physiological tests for all isolates. The production of extracellular polymeric substances was also preliminarily screened. A total of 24 luminescent isolates were obtained, with an abundance from C. agassizi specimens. All the isolates were taxonomically characterized and were related to different species of Photobacterium, with the exception of Vibrio sp. CLD11 that was from C. agassizi. The luxAB gene was detected in about the 90% of the analysed strains.