Aims The goal of this study was to isolate and characterize a lactic acid bacteria (LAB) from donkey milk with potential beneficial properties. Methods and Results Lactic acid bacteria were isolated from donkey milk and identified based on physiological, biochemical and molecular methods. The isolate that presented highest bacteriocin potential (Lactobacillus plantarum LP08AD) was evaluated for the production of bacteriocin, including stability in the presence of various enzymes, surfactants, salts, pH and temperatures. Bactericidal effect of bacteriocin LP08AD on Listeria monocytogenes, Enterococcus faecium and Lactobacillus curvatus was shown for actively growing and stationary cells. Similar growth and bacteriocin production were observed when strain LP08AD was cultured in MRS broth at 30 degrees C or 37 degrees C. Bacteriocin LP08AD adhered at low levels on the producer cells (200AUml1). The presence of plantaricin W gene on the genomic DNA was recorded based on PCR. Good growth for strain LP08AD was recorded in MRS broth with pH from 5 center dot 0 to 9 center dot 0 and LP08AD grew well in the absence of oxbile or concentration below 0 center dot 8%. Lact.plantarum LP08AD was applied to the small intestinal epithelial polarized monolayers of H4, PSIc1 and CLAB and demonstrated low attachment ability on all cell lines studied, with values with a similar behaviour for cells from human and pig origin. Conclusions Bacteriocin-producing Lact.plantarum LP08AD might be useful in the design of novel functional foods with potential probiotic or biopreservation properties. Significance and Impact of the Study To the best of our knowledge, this is the first report on detection and characterization of bacteriocinogenic Lact.plantarum from donkey milk. The strain LP08AD shows to have potential beneficial properties, as demonstrated by the use of noncancerogenic cell lines.