Lactobacilli represent a wide range of bacterial species with several implications for the human host. They play a crucial role in maintaining the ecological equilibrium of different biological niches and are essential for fermented food production and probiotic formulation. Despite the consensus about the 'health-promoting' significance of Lactobacillus genus, its genotypic and phenotypic characterization still poses several difficulties. The aim of this study was to assess the integration of different approaches, genotypic (16S rRNA gene sequencing), proteomic (MALDI-TOF MS) and metabolomic (1H-NMR), for the taxonomic and metabolic characterization of Lactobacillus species. For this purpose we analyzed 40 strains of various origin (intestinal, vaginal, food, probiotics), belonging to different species. The high discriminatory power of MALDI-TOF for species identification was underlined by the excellent agreement with the genotypic analysis. Indeed, MALDI-TOF allowed to correctly identify 39 out of 40 Lactobacillus strains at the species level, with an overall concordance of 97.5%. In the perspective to simplify the MALDI TOF sample preparation, especially for routine practice, we demonstrated the perfect agreement of the colony-picking from agar plates with the protein extraction protocol. 1H-NMR analysis, applied to both culture supernatants and bacterial lysates, identified a panel of metabolites whose variations in concentration were associated with the taxonomy, but also revealed a high intra-species variability that did not allow a species-level identification. Therefore, despite not suitable for mere taxonomic purposes, metabolomics can be useful to correlate particular biological activities with taxonomy and to understand the mechanisms related to the antimicrobial effect shown by some Lactobacillus species.