In this study, Lactococcus lactis subsp. hordniae JNU533 (JNU533) was isolated from Swiss-type cheese, and the bacteriocin produced by this strain was characterized. The spot-on-lawn assay was used to determine the antimicrobial spectrum and characteristics of the JNU533-derived bacteriocin. Results confirmed that the JNU533-derived bacteriocin inhibited the growth of lactic acid bacteria. The size of the bacteriocin was approximately 4.9 kDa, and it was heat- and pH-stable under various temperature and pH conditions. Furthermore, the possibility of using JNU533 as a starter culture in the manufacturing of fermented dairy products was assessed. A single colony of JNU533 was inoculated into 10% skim milk containing 0.5% glucose to investigate its characteristics in milk culture. The decrease in the pH was similar to that elicited by Lactobacillus delbrueckii subsp. bulgaricus. Furthermore, the results confirmed that JNU533 inhibited the growth of various bacteria and could be used as a milk fermentation starter. This study highlights the characteristics of the bacteriocin produced by JNU533 and the growth features of this strain in a skim milk medium.