Due to a large adaptability to different cultivation conditions and limited input compared to other cereals, sorghum is considered an emerging crop. Its antioxidant properties, high fiber content and low glycemic index also make it a valuable addition to a healthy diet, nevertheless, the presence of antinutritional factors and the lack of gluten, hamper its use as food ingredient. This study investigated the impact of sourdough fermentation on sorghum nutritional quality. Lactic acid bacteria dominating sorghum flour and sourdough were identified by culture-dependent analysis revealing Lactiplantibacillus plantarum as the dominant species found in the mature sourdough, whereas Weissella cibaria and Weissella paramesenteroides were the species isolated the most after the first refreshment. Among yeasts, Saccharomyces cerevisiae was the most prevalent. Lactic acid bacteria pro-technological and functional performances as starter were evaluated in sorghum type-II sourdoughs through an integrated characterization combining chromatographic and NMR spectroscopic techniques. The metabolic profile of the strains mainly grouped together W. cibaria strains and W. paramesenteroides AI7 which distinguished for the intense proteolysis but also for the presence of compounds particularly interesting from a physiological perspective (allantoin, glutathione, γ-aminobutyric acid and 2-hydroxy-3-methylbutyric acid), whose concentration increased during fermentation in a species or strain specific matter. •Lactic acid bacteria and yeasts were isolated from sorghum flour and sourdough.•Lactiplantibacillus plantarum was dominant in the mature sourdough.•LAB pro-technological and functional performances were evaluated.•Metabolome was investigated through chromatographic and NMR techniques.•W. cibaria and W. paramesenteroides strains caused an intense proteolysis.