Donkey milk is known for its beneficial aspects such as low fat and cholesterol, total protein and casein content, high lactose, whey protein, calcium, selenium and vitamin D3 content. It also has nutraceutical and utilitarian properties that can boost invulnerability, regulate digestion and conveniently change gut microbiota. The current study was focused on the effect of probiotic bacterial metabolites from donkey milk against human bacterial pathogens. The effective isolate VITRAAJ14 was found to be non-hemolytic and was tolerant to acid, NaCl and bile salt. Antibacterial activity by cross streak method showed maximum zone of inhibition. Well diffusion method also exhibited effective inhibition against Escherichia coli (MTCC No. 9721), Staphylococcus aureus (MTCC No. 3160) and Salmonella enterica (MTCC No. 8587) with fractions obtained from chloroform: ethyl acetate. The metabolites capable of inhibiting pathogens were identified as oleyl alcohol by Gas chromatography- mass spectrometry (GCMS). Lead compounds detected in GCMS was studied for its functional group by Fourier transform infrared spectroscopy (FTIR), which revealed the presence of alcoholic stretch, carboxylic acid OH stretch, alkene and amide bond. The isolate VITRAAJ14 was identified to be the closest neighbour of Bacillus species (MH100985) by 16S rRNA gene sequencing. To our knowledge, current study is the first-time report on the presence of Bacillus sp. from donkey milk showing antibacterial activity due to its ability to produce oleyl alcohol. Scanning electron microscopic images revealed the presence of rod-shaped morphology of the effective bacterium VITRAAJ14.