The possibility of muropeptides obtaining of peptidoglycans of Lactobacillus delbrueckii subsp. Bulgaricus B-3964 cell walls by the combination of the use of autolytic processes and enzyme treatment of biomass with the participation of lysozyme and papain has been considered. It has been established that the most significant autolytic changes in biomass occur in the application of high-temperature processing (90°C for 30 minutes) in the final stage of the logarithmic phase of bacterial growth. Thus, after eighth hour of biomass incubation at 37°C, the amino acid content in the culture medium was 1.8 mg/cm3, and at 90°C it was 5.7 mg/cm3. In order to further destruction of biomass autolysate and obtaining of low molecular weight peptidoglycan fragments, the process of its enzymatic hydrolysis was studied with lysozyme and papain separately and at their combination. The highest content of low molecular weight peptides in the reaction medium occurred at enzymatic hydrolysis of biomass Lactobacillus delbrueckii subsp. Bulgaricus B-3964 by the composition of enzymes at a ratio of lysozyme : papain 1:2. At a concentration of enzymes 10 mg/cm3, the content of low molecular weight peptides was 7.2 mg/cm3 after eighth hour of incubation of the reaction mixture. The results of studies have been shown that the efficiency of enzymatic hydrolysis of autolysates is much higher. Thus, the amount of low molecular weight peptides in the hydrolysate obtained by processing the autolysate with the composition of lysozyme : papain 1:2 at an enzymes concentration 10 mg/cm3 and the duration of the process for 8 hours by 36% higher than for similar hydrolysis parameters without the use of the process of autolysis.The method of gel chromatography was proved that in the hydrolysate there are fractions of protein compounds with a molecular weight in the range of 70–90 kDa, 30–40 kDa and 294–650 Da. The molecular weight of the latter fraction corresponds to the mass of the muramyl dipeptide. The presence of muropeptides was proved by reaction with the Anthron reagent.