The emergence of nearly untreatable infections caused by multidrug-resistant bacteria has led to a new public health concern in which a need for development of alternative non-antibiotic strategies has become urgent. The activity of metabolites produced by new strains of Lactobacillus against multidrugresistant bacteria was investigated. The objective of this work was to isolate and identify lactobacilli from artisanal kefir by 16S rRNA gene sequencing as well as to evaluate the effect of the growth of Lactobacillus species in the Man Rogosa and Sharpe broth, supplemented with Tween 80 at concentrations of 0.6, 0.4, and 0.2 %. Cell Free Supernatants (CFSs) were obtained from these lactobacilli cultures and with them, organic acids (lactic acid and acetic acid) and ethanol were quantified by HPLC followed by the determination of their antimicrobial activities against eight strains of Staphylococcus aureus-MRSA and Klebsiella pneumonia KPC strains. The GenBank BLAST analysis revealed that, the isolated lactobacilli belonged to Lactobacillus paracasei (n=4) and Lactobacillus plantarum (n=5) strains. Increasing concentrations of Tween 80 did not affect the growth of Lactobacillus species significantly when compared to their controls (MRS broth). HPLC analysis of CFSs showed concentrations greater than 18.0, 4.0 and 1.0 g/L for lactic acid, acetic acid and ethanol, respectively. All CFSs were able to inhibit all pathogenic microorganisms evaluated. The percentage of inhibition was on average greater than 88% for MRSA and KPC strains. The antimicrobial activity was dependent on the CFSs tested. Based on these experimental conditions, organic acids and ethanol are likely to be responsible for this antimicrobial activity.