<p>Kefir is a probiotic and a rich source of bioactive compounds including antimicrobial peptides. Due to its natural versatility and characteristics, peptides prospected from kefir may become an affordable and sustainable tool against microorganisms. Here, we investigated the peptide profile and evaluate the antimicrobial, membrane permeabilization, and antibiofilm properties of a peptide-rich fraction obtained from a bovine milk-based kefir from Brazil. The milk-fermented drink was centrifuged using a three-step ultrafiltration process. The peptide profile of the resulting fraction (&lt; 3&#xa0;kDa) was analyzed by uHPLC and MALDI-TOF MS-Spectra. The antimicrobial activity of the peptide fraction was assessed using the broth microdilution technique. Quantitative assays and qualitative fluorescence microscopy images were used to assess its effects on bacterial membrane and antibiofilm formation. The peptide profile analysis of the fraction led to the discovery of 32 peptide molecules, with sizes ranging from 700 to 1400&#xa0;Da. The combination of these peptides exhibited broad-spectrum bacteriostatic and bactericidal activity. The Sytox Green assay suggested that these peptides may exert their antimicrobial mechanisms via membrane damage. The peptide fraction also inhibited the biofilm formation of <i>Staphylococcus</i> <i>aureus</i> MRSA up to 69.9 to 81.4%. This study unraveled the antimicrobial potential of a peptide fraction from a Brazilian milk-based kefir. It is important to note that, to our knowledge, this is the first report that identifies peptides from kefir beverage as inhibitors of bacterial biofilm formation. These results emphasize this product as a source of new biomolecules that may be explored as biotechnological alternatives to combat microorganisms.</p>