The purpose of this work was to examine the antimicrobial activity derived from the lactic acid bacterium, UK-3 isolated from the vaginas of women of childbearing age. Various physiological and biochemical properties of this strain were characterized. Both the BIOLOG system and phylogenetic analysis using 16S rRNA sequencing were utilized for identification, and the strain was designated as Lactobacillus plantarum UK-3, and registered in GenBank as [JK266589]. Growth rate, production of organic acids (e.g., lactic acid and acetic acid), and pH during growth were monitored. The maximum concentrations of lactic acid and acetic acid were approximately 684.11 mM and 174.26 mM, respectively, and pH changed from 7.0 to 3.7 after 72 h of incubation. High performance liquid chromatography was used to confirm lactic acid and acetic acid production. Significant antimicrobial activity of the concentrated supernatant was demonstrated against various Gram-positive (e.g., Staphylococcus aureus, Staphylococcus epidermidis, Methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, Neisseria species., Listeria monocytogenes), Gram-negative bacteria (e.g., Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis), and yeast (e.g., Candida albicans) by the plate diffusion method. As a result, the concentrated L. plantarum UK-3 cultures had lower acidity and inhibited the growth of all microorganisms tested, whereas the growth of L. acidophilus was not affected.