Lacticin NK34 is a small nisin-like bacteriocin present in the supernatants of an isolate of Lactococcus lactis from jeotgal, a salted and fermented Korean food made with seafood such as shrimp, oysters, and fish. Recently, we demonstrated that a partially purified NK34 is highly effective against various Staphylococcus species in a murine infection model. In this study, the two major bacterial pathogens associated with bovine mastitis, Streptococcus aureus and S. agalactiae, were evaluated for their susceptibility to NK34 in vitro using a standard teat-dip assay as well as in vivo using mastitic cows infected with one of these pathogenic strains. The experimental analyses showed a significant decrease (up to 98 times) in the bacterial numbers between the NK34-treated and -untreated teats. Moreover, a dramatic reduction in somatic cell counts was observed at 3 days post-treatment with 10 ml of NK34 administered directly into the mastitic cows. Neither S. aureus nor S. agalactiae were recovered. Taken together, these results imply that lacticin NK34 is an alternative antimicrobial substitute for the treatment of bovine mastitis, caused especially by either S. aureus or S. agalactiae.