[Objective] Inhibition of lactobacilli metabolites on Streptococcus pyogenes was analyzed. [Methods] Three lactobacilli strains was grown in MRS broth overnight, then the culture supernatants were collected and evaluated for inhibitory activity. Inhibition was determined by measuring inhibition zone diameters with double plate punching method. Then HPLC and 4-Aminoantipyrine were used to determine the concentration of organic acids and H2O2 respectively in lactobacilli metabolites. Finally, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of lactic acid, acetic acid and H2O2 on S. pyogenes were determined. [Results] Lactobacillus plantarum KLDS1.0667 had the strongest inhibitory effect among the three strains, followed by L. paracasei KLDS1.0342-1 and L. helveticus KLDS1.0203. L. plantarum KLDS1.0667 had the highest production of lactic acid and acetic acid, followed by KLDS1.0342-1 and KLDS1.0203. However, L. helveticus KLDS1.0203 had the highest production of H2O2, followed by KLDS1.0667 and KLDS1.0342-1. The diameters of inhibition zone all decreased after elimination of H2O2 in lactobacilli supernatants, and no obvious inhibition zones were detected when the pH of lactobacilli supernatants was adjusted to 7.0. The results indicated that organic acids and H2O2 were the main inhibiting substances in lactobacilli metabolites, further lactic acid was the most important inhibiting substance. MIC of lactic acid, acetic acid and H2O2 on Streptococcus pyogenes were 1.28, 0.64 and 0.008 g/L, respectively. MBC of lactic acid, acetic acid and H2O2 on Streptococcus pyogenes were 5.12, 2.56 and 0.032 g/L, respectively. [Conclusion] The metabolites of lactobacilli could inhibit the growth of Streptococcus pyogenes, and the organic acids and H2O2 were the main inhibiting substances.