This study aimed to identify phenolic acids and flavonoids in the red sorghum variety PAN 3860 and to determine changes in their concentrations during fermentation with lactobacilli. Sorghum sourdoughs fermented with two binary strain combinations, Lactobacillus plantarum and Lactobacillus casei or Lactobacillus fermentum and Lactobacillus reuteri , were compared to chemically acidified controls. Four glycerol esters were tentatively identified, caffeoylglycerol, dicaffeoylglycerol, coumaroyl-caffeoylglycerol, and coumaroyl-feruloylglycerol, that have previously not been detected in sorghum. Chemical acidification resulted in hydrolysis of phenolic acid esters and flavonoid glucosides. During lactic fermentation, phenolic acids, phenolic acid esters, and flavonoid glucosides were metabolized. Analysis of ferulic acid, caffeic acid, and naringenin-glucoside contents in single-strain cultures of lactobacilli demonstrated that glucosidase, phenolic acid reductase, and phenolic acid decarboxylase activities contributed to polyphenol metabolism. This study demonstrates that microbial fermentation of sorghum affects the content of polyphenols and can influence the nutritional value and antimicrobial activity of sorghum.