The strain HM02-04 isolated from human milk was able to produce the antimicrobial substance (AMS) against vancomycin-resistant enterococci (VRE) which causes a serious infection. Based on its morphology, biochemical character, 16S rDNA nucleotide sequence and MALDI-TOF MS analysis, it was identified as Enterococus hirae. Partial purification by ammonium sulphate precipitation provided the active AMS which was sensitive to proteolytic enzymes, pepsin, trypsin, proteinase K, and proteinase E. It was elucidated that the AMS obtained had a proteinaceous structure. Partial purified bacteriocin (PPB) obtained by ammonium sulphate precipitation showed thermal stability when heated at 121 °C for 15 min and pH stability of 3.0-9.0. This PPB had a narrow spectrum only against the VRE group. It exhibited various inhibition activities of 800-3200 AU/ml against 10 isolates of VRE which had different antibiotic susceptibility spectrum. To enhance its inhibition activity against VRE, various organic acids were investigated and their results showed that 50 mM formic acid produced 100% growth inhibition against VRE 426 of 6.9 log CFU/ml while the low concentration of 5 mM did not show growth inhibition activity. However, the combination of 1600 AU/ml PPB and 5 mM formic acid could enhance the growth inhibition activity against VRE 426 at 7 log CFU/ml by 31% which was approximately 2 folds higher than the formic acid treatment alone. In addition, the mode of bactericidal activity was pore formation at bacterial cell wall, as confirmed by scanning electron microscopy analysis. Its mode of action was also similar to that of the treatment with either formic acid or PPB alone. The results suggested that the combination of bacteriocin PPB produced by E. hirae HM02-04 and formic acid has a great potential as a disinfectant via the inhibition of the growth of VRE in the hospital.