Using the Gram-positive Staphylococcus aureus as the indictor bacterium, fourteen antibacterial strains were initially obtained by the bilayer-media screening method from the raw milk samples, and one isolate was found to exhibit the higher antibacterial activity against the indicator. This isolate was further studied on its individual and cultural morphology features, partial physiological and biochemical reaction activities, G+C content, the sequence features of the 16S rDNA and the species-specific N-acetylmuraminidase gene (acmA), consequently, it was identified as the Lactococcus lactis subsp. lactis strain, named as MB191. An evaluation of the antimicrobial spectra of MB191 was subsequently performed, it showed the remarkable activities against not only the tested Gram-positive bacteria, but also several Gram-negative bacteria including Pseudomonas syringae and P. fluorescens, as well as the yeast Debaryomyces hansenii, which was a distinctive feature that was not reported prior to this study.