According to the world health organization report from September 2016, the development of pathogenic bacteria resistance to antimicrobial drugs is one of the most important problems of the modern medicine. In this regard, the urgent task is the search for alternative antibiotics for the treatment of bacterial infections. One approach to solving this problem is obtaining antimicrobial compounds synthesized by probiotic lactic acid bacteria. The probiotic strain of Lactobacillus plantarum 8P-A3, was chosen to study its antimicrobial action. This strain produces at least two bacteriocins - plantaricin EF and plantaricin NC8. The chromatographic isolation of peptide fractions from the supernatant was carried out using a polymer sorbent based on methacrylic acid and ethyleneglycol dimethacrylate. Optimal parameters for chromatographic process were determined. It is shown that all the target biologically active substances were bound with the sorbent in sorption at acidic pH values. Elution was performed in isocratic mode. The antimicrobial activity of the obtained peptide fractions against indicator culture was determined by turbidimetric method. During incubation process, the turbidity of the microbial suspension was determined by measuring the optical density at λ = 600 nm. It is revealed that the fraction obtained at rinse by eluent with pH 8 has the maximum inhibitory ability. Chromatomass-spectrometry analysis of the peptide fraction was carried out using Shimadzu LCMS-8040. The antimicrobial activity of the fraction is comparable to the action of ampicillin against gram-negative bacteria Escherichia coli. To confirm the peptide nature of the antimicrobial activity of the fraction, an indicator culture was incubated with the fraction treated with proteolytic enzymes (trypsin). It is determined the fraction can be stored at -18 °C and saves antimicrobial properties after defrosting.