In mollusk aquaculture, a large number of Vibrio species are considered major pathogens. Conventional methods based on DNA amplification and sequencing used to accurately identify Vibrio species are unsuitable for monitoring programs because they are time-consuming and expensive. The aim of this study was, therefore, to develop the MALDI-TOF MS method in order to establish a rapid identification technique for a large panel of Vibrio species. We created the EnviBase containing 120main spectra projections (MSP) of the Vibrio species that are potentially responsible for mollusk diseases, comprising 25 species: V. aestuarianus, V. cortegadensis, V. tapetis and species belonging to the Coralliilyticus, Harveyi, Mediterranei, and Orientalis clades. Each MSP was constructed by the merger of raw spectra obtained from three different media and generated by three collaborating laboratories to increase the diversity of the conditions and thus obtain a good technique robustness. Perfect discrimination was obtained with all of the MSP created for the Vibrio species and even for very closely related species as V. europaeus and V. bivalvicida. The new EnviBase library was validated through a blind test on 100 Vibrio strains performed by our three collaborators who used the direct transfer and protein extraction methods. The majority of the Vibrio strains were successfully identified with the newly created EnviBase by the three laboratories for both protocol methods. This study documents the first development of a freely accessible database exclusively devoted to Vibrio found in marine environments, taking into account the high diversity of this genus.