The genus Vibrio currently contains 147 recognized species widely distributed, including pathogens for aquatic organisms. Vibrio infections in elasmobranchs are poorly reported, often with identifications as Vibrio sp. and without detailed diagnostic insights. The purpose of this paper is the description of the isolation and identification process of Vibrio spp. following a mortality event of Scyliorhinus canicula juvenile reared in an Italian public aquarium. Following investigations aimed at excluding the presence of different pathogens of marine fish species (parasites, bacteria, Betanodavirus), several colonies were isolated and subjected to species identification using the available diagnostic techniques (a biochemical test, MALDI-TOF MS, and biomolecular analysis). Discrepancies were observed among the methods; the limits of biochemistry as a unique tool for Vibrio species determination were detected through statistical analysis. The use of the rpoB gene, as a diagnostic tool, allowed the identification of the isolates as V. crassostreae and V. cyclotrophicus. Although the pathogenic role of these microorganisms in lesser-spotted dogfish juveniles has not been demonstrated, and the presence of further pathogens cannot be excluded, this study allowed the isolation of two Vibrio species in less-studied aquatic organisms, highlighting the weaknesses and strengths of the different diagnostic methods applied.