The identification of microorganisms in vinegar production remains a challenge, mainly, due to the difficulty of isolating and characterizing the microbiota involved. Metaproteomics reveals microbial diversity and behavior by analysis of proteins, offering precise insights into key moments and conditions in biological processes like acetification. This study consists of a thorough metaproteomic analysis during the elaboration of a new vinegar, Verdejo wine vinegar. Acetic acid fermentation occurred by submerged culture in an 8L automated Frings reactor operated in semi-continuous mode. LC/MS-MS identified 1,626 proteins from 149 taxonomic groups, mainly Acetobacteraceae (1,529), followed by yeast (47), lactic acid bacteria (29), and Archaea (21). Komagataeibacter (73.7%), Acetobacter (10.7%), Gluconacetobacter (1.7%), Novacetimonas (1.6%), and Gluconobacter (1.3%) were prevalent genera, with K. europaeus being the dominant species (56.7%). To our knowledge, this is the first metaproteomic approach to comprehensively address the study of so diverse microbial populations using an innovative protein processing adaptation for LC-MS/MS. GO Term analysis emphasized "heterocyclic compound binding" and "organic substance metabolic process", while protein-protein interaction analysis, BCAA biosynthesis, TCA cycle, and stress response mechanisms. Overall, this study aims to characterize the microbiota driving Verdejo wine acetification and explore its composition, functions, and key metabolic processes.