The identification of bacteria by means of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry directly using whole cells has become a standard method in clinical diagnosis due to its rapidity and simplicity. Nevertheless, the analysis of environmental samples with this approach still represents a challenge due to the enormous microbial diversity existing on earth and the lack of a comprehensive database. Most of the environmentally relevant species comprise only one unique strain, while pathogens such as Escherichia coli, with 667 described strains, are well documented. In such case, identification of the proteins responsible for the peak signals within MALDI-TOF spectra can give crucial information for species discrimination. To give higher confidence in MALDI-TOF biomarker description we exploited information from proteins identified by shotgun nanoLC-MS/MS, consisting of the identification and quantification of low-molecular-weight proteins after SDS-PAGE, in-gel trypsin proteolysis and analysis of tryptic peptides. We also proposed the standardization of the inclusion of internal calibrants in the bacterial sample to improve the accuracy of the MALDI-TOF measurements. In this way, nine candidate biomarkers were tentatively proposed for Ruegeria lacuscaerulensis ITI-1157. The conserved biomarkers were theoretically deduced for all other Ruegeria strains whose genomes have been sequenced and their corresponding m/z MALDI-TOF signals were estimated. Among these, DNA-binding protein, HU, and ribosomal proteins, L29, L30, L32 and S17, were shown experimentally to be also the most prominent and conserved signals in the other strain tested, Ruegeria pomeroyi DSS-3. Thus, we suggested that these five biomarkers, which give rise to 10 m/z peak signals derived from the mono- and doubly protonated proteins, are the best candidates for identifying bacteria belonging to the Ruegeria genus, and quickly assessed their phylogenetic proximity to described species. As an application of these biomarkers, we quickly screened 30 seawater bacterial isolates by MALDI-TOF and found one belonging to the Ruegeria genus, as further confirmed by 16S RNA sequencing. Due to its simplicity and effectiveness, this technique could be of immense value in monitoring bacteria in the environment in the near future.