Due to their high salt and alkali tolerance mesophilic bacteria of the genus Vibrio are widely spread in both marine and fresh water. Furthermore, they are one of the main causes for diarrheal diseases elicited via contaminated water or raw seafood. Due to global warming, involving an increase in water temperature an increase of potentially pathogenic Vibrio spp. is also expected in Northern Europe. Conventional methods for a reliable identification of microorganisms, such as analyses of 16S rRNA sequence analyses seem to be unreliable in case of Vibrio. As a more reliable alternative, the identification of Vibrio by MALDI-TOF mass spectrometry has proven. The principle is based on the generation of a significant mass spectrum according to the protein composition of a bacterial cell. This spectrum is compared to reference spectra in a database and leads depending on the quality of the database to the identification. To date, the identification via MALDI could only be performed in pure cultures with a few exceptions. Consequently, the aim of this study was to analyze bacterial mixtures of different complexity with regard to Vibrio spp. Species specific biomarkers were screened which are only detectable for investigated strain. Furthermore, it was evaluated if environmental samples can be analyzed using these biomarkers with regard to Vibrio spp. without prior isolation. In this study, it was shown that MALDI-TOF is a reliable method for the identification of Vibrio species. On the basis of genus and species specific biomarkers (Erler et al., 2014), which served as basis for identification at the species level in the present study, examined pure cultures were successfully identified. Furthermore, associated biomarkers were detected in analyzed mixtures that have contributed for at least one species to successful identification. Even in mixtures with different mixing ratios biomarkers for each species were identified that exhibited a lower ratio and subsequently a lower cell mass. In the investigated environmental samples Vibrio species were also detected, using defined biomarkers although the microbial composition was unknown. However, the analysis was complicated by partially defined same mass-to-charge ratios of biomarkers. Consequently, it was questioned to what extent previously identified biomarkers can be confirmed. Further studies need to be performed to implement a method which withstands a reliable validation. This study, however, demonstrated that the identification on basis of specific biomarkers for Vibrio species is a promising approach. Further analyses will benefit using MALDI particularly within the scope of the risk assessment for potentially pathogenic Vibrio species.